U.S. patent application number 15/633126 was filed with the patent office on 2017-10-26 for crispr having or associated with destabilization domains.
The applicant listed for this patent is The Broad Institute Inc., Massachusetts Institute of Technology, President and Fellows of Harvard College. Invention is credited to Amit Choudhary, Bernd Zetsche, Feng Zhang.
Application Number | 20170306307 15/633126 |
Document ID | / |
Family ID | 55273518 |
Filed Date | 2017-10-26 |
United States Patent
Application |
20170306307 |
Kind Code |
A1 |
Zhang; Feng ; et
al. |
October 26, 2017 |
CRISPR HAVING OR ASSOCIATED WITH DESTABILIZATION DOMAINS
Abstract
The disclosure includes non-naturally occurring or engineered
CRISPR Cas9, each associated with at least one destabilization
domain (DD), along with compositions, systems and complexes
involving the DD-CRISPR Cas9, nucleic acid molecules and vectors
encoding the same, delivery systems involving the same, uses
therefor.
Inventors: |
Zhang; Feng; (Cambridge,
MA) ; Zetsche; Bernd; (Gloucester, MA) ;
Choudhary; Amit; (Cambridge, MA) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
The Broad Institute Inc.
Massachusetts Institute of Technology
President and Fellows of Harvard College |
Cambridge
Cambridge
Cambridge |
MA
MA
MA |
US
US
US |
|
|
Family ID: |
55273518 |
Appl. No.: |
15/633126 |
Filed: |
June 26, 2017 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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PCT/US2015/067177 |
Dec 21, 2015 |
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15633126 |
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62096656 |
Dec 24, 2014 |
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62181151 |
Jun 17, 2015 |
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Current U.S.
Class: |
1/1 |
Current CPC
Class: |
C12N 15/102 20130101;
A01K 2207/10 20130101; C12N 15/907 20130101; A01K 67/0275 20130101;
C12Y 105/01003 20130101; A01K 2217/05 20130101; C12N 9/22 20130101;
A61K 48/005 20130101; C07K 2319/095 20130101; A01K 2227/105
20130101; C12N 9/003 20130101; C07K 2319/00 20130101; C07K 14/721
20130101 |
International
Class: |
C12N 9/22 20060101
C12N009/22; A01K 67/027 20060101 A01K067/027; C07K 14/72 20060101
C07K014/72; A61K 48/00 20060101 A61K048/00; C12N 15/90 20060101
C12N015/90; C12N 9/06 20060101 C12N009/06 |
Goverment Interests
STATEMENT AS TO FEDERALLY SPONSORED RESEARCH
[0002] This invention was made with government support under grant
nos. MH100706 and MH110049 awarded by the National Institutes of
Health. The government has certain rights in the invention.
Claims
1. A composition comprising a non-naturally occurring or engineered
CRISPR Cas9 associated with or fused to at least one
destabilization domain (DD)
2. The composition of claim 1, wherein the Cas9 comprises an Sp
Cas9, an Sa Cas9, an St Cas9, or an Fn Cas9.
3. The composition of claim 1 or 2, wherein the Cas9 comprises a
Rec2 or HD2 truncation.
4. The composition of claim 3, wherein the truncation comprises
removal or replacement with a linker.
5. The composition of claim 4, wherein the linker comprises a
branch or otherwise allows for tethering of the DD and/or a
functional domain.
6. The composition of claim 1, wherein the DD is associated with
the CRISPR Cas9 by fusion with said CRISPR Cas9.
7. The composition of claim 6, which comprises at least one DD
fused to the N-terminus or C-terminus of the CRISPR Cas9.
8. The composition of claim 7, comprising at least two DDs and
wherein a first DD is fused to the N-terminus of the CRISPR Cas9
and a second DD is fused to the C-terminus of the CRISPR Cas9, the
first and second DDs being the same or different.
9. The composition of claim 6, wherein the fusion comprises a
linker between the DD and the CRISPR Cas9.
10. The composition of claim 9, wherein the linker comprises,
consists essentially of or consists of: GlySer linker; or a
localization signal.
11. The composition of claim 1, further comprising at least one
Nuclear Export Signal (NES) or at least one Nuclear Localization
Signal (NLS).
12. The composition of claim 11, wherein the Cas9 comprises two or
more NESs.
13. The composition of claim 7, wherein one of the at least one DD
comprises ER50 or DHFR50.
14. The composition of claim 1, wherein the Cas9 comprises at least
one mutation.
15. The composition of claim 14, wherein the Cas9 comprises a
nickase.
16. The composition of claim 15, wherein the Cas9 comprises
Staphylococcus aureus Cas9 (SaCas9) and the mutation comprises
N580A.
17. The composition of claim 14, wherein the Cas9 has substantially
no nuclease activity due to the mutation(s).
18. The composition of claim 1, wherein the Cas9 comprises a split
Cas9.
19. The composition of claim 1, wherein the Cas9 comprises a
functional domain.
20. A polynucleotide encoding the CRISPR Cas9 and associated or
fused DD of claim 1.
21. The polynucleotide of claim 20, wherein the encoded CRISPR Cas9
and associated DD are operably linked to a first regulatory
element.
22. The polynucleotide of claim 20, wherein a DD is encoded and is
operably linked to a second regulatory element.
23. The polynucleotide of claim 21, wherein the first regulatory
element comprises a promoter and optionally comprises an
enhancer.
24. The polynucleotide of claim 22, wherein the second regulatory
element comprises a promoter and optionally comprises an
enhancer.
25. The polynucleotide of claim 21, wherein the first regulatory
element comprises an early promoter.
26. The polynucleotide of claim 22, wherein the second regulatory
element is a late promoter.
27. The polynucleotide of claim 22, wherein the second regulatory
element comprises an inducible control element, optionally the tet
system, or a repressible control element, optionally the tetr
system.
28. A vector(s) comprising the polynucleotide(s) of claim 20.
29. The vector(s) of claim 28, comprising one or more plasmid or
viral vector.
30. A cell or cell line or non-human animal, or progeny thereof,
modified so as to contain the composition of claim 1 or a
polynucleotide of claim 20 or a vector(s) of claim 28.
31. The non-human animal of claim 30, which constitutively
expresses the CRISPR Cas9-DD fusion.
32. The non-human animal of claim 31, wherein the non-human animal
is a mouse.
33. Progeny of the cell or cell line or non-human animal of claim
30.
34. A DD-CRISPR-Cas System comprising a composition of claim 1, a
polynucleotide of claim 20, or a vector of claim 28.
35. A method of controlled targeting of a polynucleotide of
interest in a cell comprising a DD-CRISPR-Cas9 system of claim 34
present in the cell whereby a guide polynucleotide of the DD-CRISPR
Cas9 system targets the polynucleotide of interest; and having, in
a controlled manner, a stabilizing ligand as to the DD present in
the cell.
36. A method of treatment of a subject in need thereof comprising
administering a DD-CRISPR-Cas9 system of claim 34 to the subject
whereby it is present in cell(s) of the subject whereby a guide
polynucleotide of the DD-CRISPR Cas system targets a polynucleotide
of interest involved in a condition of the subject, the targeting
of which results in a treatment therefor; and having, in a
controlled manner, a stabilizing ligand as to the DD present in
cell(s) of the subject.
37. A CRISPR-Cas9 complex comprising the composition of claim 1, a
guide polynucleotide and a nucleic acid target.
Description
RELATED APPLICATIONS AND INCORPORATION BY REFERENCE
[0001] This application is a continuation-in-part to International
patent application Serial No. PCT/US2015/067177 filed Dec. 21, 2015
and published as PCT Publication No. WO 2016/106244 on Jun. 30,
2016 and which claims priority from U.S. application Ser. No.
62/096,656, filed Dec. 24, 2014, and U.S. application Ser. No.
62/181,151, filed Jun. 17, 2015.
[0003] Each of these patents, patent publications, and
applications, and all documents cited therein or during their
prosecution ("appln cited documents") and all documents cited or
referenced in the appln cited documents, together with any
instructions, descriptions, product specifications, and product
sheets for any products mentioned therein are incorporated by
reference herein, and may be employed in the practice of the
invention. Moreover, all documents cited or referenced herein
("herein cited documents"), and all documents cited or referenced
in herein cited documents, together with any manufacturer's
instructions, descriptions, product specifications, and product
sheets for any products mentioned herein or in any document
incorporated by reference herein, are hereby incorporated herein by
reference, and may be employed in the practice of the invention.
All referenced documents are incorporated by reference to the same
extent as if each individual document was specifically and
individually indicated to be incorporated by reference.
SEQUENCE LISTING
[0004] The instant application contains a Sequence Listing which
has been submitted electronically in ASCII format and is hereby
incorporated by reference in its entirety. Said ASCII copy, created
on Mar. 17, 2016, is named 47627992010_SL.txt and is 114 bytes in
size.
FIELD OF THE INVENTION
[0005] The present invention generally relates to Clustered
Regularly Interspaced Short Palindromic Repeats (CRISPR), CRISPR
enzyme (Cas9), CRISPR-Cas9 or CRISPR system or CRISPR-Cas9 complex,
components thereof, nucleic acid molecules, e.g., vectors,
involving the same and uses of all of the foregoing, amongst other
aspects.
BACKGROUND OF THE INVENTION
[0006] Recent advances in genome sequencing techniques and analysis
methods have significantly accelerated the ability to catalog and
map genetic factors associated with a diverse range of biological
functions and diseases. Precise genome targeting technologies are
needed to enable systematic reverse engineering of causal genetic
variations by allowing selective perturbation of individual genetic
elements, as well as to advance synthetic biology,
biotechnological, and medical applications. Although genome-editing
techniques such as designer zinc fingers, transcription
activator-like effectors (TALEs), or homing meganucleases are
available for producing targeted genome perturbations, there
remains a need for new genome engineering technologies that employ
novel strategies and molecular mechanisms and are affordable, easy
to set up, scalable, and amenable to targeting multiple positions
within the eukaryotic genome. This would provide a major resource
for new applications in genome engineering and biotechnology.
[0007] Citation or identification of any document in this
application is not an admission that such document is available as
prior art to the present invention.
SUMMARY OF THE INVENTION
[0008] There exists a pressing need for alternative and robust
systems and techniques for sequence targeting with a wide array of
applications. This invention addresses this need and provides
related advantages. The CRISPR/Cas or the CRISPR-Cas system (both
terms may be used interchangeably throughout this application) does
not require the generation of customized proteins to target
specific sequences but rather a single Cas enzyme can be programmed
by a short RNA molecule to recognize a specific DNA target, in
other words the Cas enzyme can be recruited to a specific DNA
target using said short RNA molecule. It will be appreciated that
reference herein to the Cas protein is restricted to Cas9,
including SpCas9, SaCas9 and other orthologs.
[0009] Adding the CRISPR-Cas system to the repertoire of genome
sequencing techniques and analysis methods may significantly
simplify the methodology and accelerate the ability to catalog and
map genetic factors associated with a diverse range of biological
functions and diseases. To utilize the CRISPR-Cas system
effectively for genome editing without deleterious effects, it is
critical to understand aspects of engineering and optimization of
these genome engineering tools, which are aspects of the claimed
invention. In some embodiments, the terms `CRISPR enzyme` and
`nucleic acid-targeting effector protein` may be used
interchangeably. Indeed, these terms and `effector protein` may
also be used interchangeably. The terms `CRISPR Cas` or `CRISPR Cas
system` and `nucleic acid-targeting system` may be used
interchangeably. The terms `CRISPR complex` and `nucleic
acid-targeting complex` be used interchangeably. Where reference is
made herein to a `target locus,` for example a target locus of
interest, then it will be appreciated that this may be used
interchangeably with the phrase `sequences associated with or at a
target locus of interest.`
[0010] In one aspect, the invention provides a non-naturally
occurring or engineered CRISPR enzyme associated with at least one
destabilization domain (DD); and, for shorthand purposes, such a
non-naturally occurring or engineered CRISPR enzyme associated with
at least one destabilization domain (DD) is herein termed a
"DD-CRISPR enzyme". In one aspect, the invention provides an
engineered, non-naturally occurring DD-CRISPR-Cas system comprising
a DD-CRISPR enzyme, wherein the CRISPR enzyme is a Cas9 protein
(herein termed a "DD-Cas9 protein", i.e., "DD" before a term such
as "DD-CRISPR-Cas9 complex" means a CRISPR-Cas9 complex having a
Cas9 protein having at least one destabilization domain associated
therewith), advantageously a type II DD-Cas9 protein, i.e., a Cas9
protein associated with at least one destabilization domain (herein
termed a "DD-Cas9 protein") and guide RNA that targets a nucleic
acid molecule such as a DNA molecule, whereby the guide RNA targets
the nucleic acid molecule, e.g., DNA molecule. The nucleic acid
molecule, e.g., DNA molecule can encode a gene product. In some
embodiments the DD-Cas9 protein may cleave the DNA molecule
encoding the gene product. In some embodiments expression of the
gene product is altered. The Cas9 protein and the guide RNA do not
naturally occur together. The invention comprehends the guide RNA
comprising a guide sequence fused to a tracr sequence. The
invention further comprehends coding for the Cas9 protein being
codon optimized for expression in a eukaryotic cell. In a preferred
embodiment the eukaryotic cell is a mammalian cell and in a more
preferred embodiment the mammalian cell is a human cell. Expression
of the gene product may be decreased. The CRISPR enzyme may form
part of a CRISPR-Cas9 system, which further comprises a guide RNA
(sgRNA) comprising a guide sequence capable of hybridizing to a
target sequence in a genomic locus of interest in a cell. In some
embodiments, the functional CRISPR-Cas9 system binds to the target
sequence. In some embodiments, the functional CRISPR-Ca9s system
may edit the target sequence, e.g., the target sequence may
comprise a genomic locus, and in some embodiments there may be an
alteration of gene expression. In some embodiments, the functional
CRISPR-Cas9 system may comprise further functional domains. In some
embodiments, the invention provides a method for altering or
modifying expression of a gene product. The method may comprise
introducing into a cell containing a target nucleic acid, e.g., DNA
molecule, or containing and expressing a target nucleic acid, e.g.,
DNA molecule; for instance, the target nucleic acid may encode a
gene product or provide for expression of a gene product (e.g., a
regulatory sequence).
[0011] The DD-CRISPR enzyme is a DD-Cas9. In some embodiments, the
DD-CRISPR enzyme is an Sp DD-Cas9. In some embodiments, the CRISPR
enzyme is an Sa DD-Cas9. In some embodiments, the CRISPR enzyme is
an St or Fn DD-Cas9, although other orthologs are envisaged. Sp and
Sa DD-Cas9s are particularly preferred, in some embodiments. In
some embodiments, the DD-CRISPR enzyme cleave both strands of DNA
to produce a double strand break (DSB). In some embodiments, the
DD-CRISPR enzyme is a nickase. In some embodiments, the DD-CRISPR
enzyme is a dual nickase. In some embodiments, the DD-CRISPR enzyme
is a deadCas9, e.g., a Cas9 having substantially no nuclease
activity, e.g., no more than 5% nuclease activity as compared with
a wild-type Cas9 or Cas9 not having had mutations to it.
[0012] In some general embodiments, the DD-CRISPR enzyme is
associated with one or more functional domains. In some more
specific embodiments, the DD-CRISPR enzyme is a deadCas9 and/or is
associated with one or more functional domains.
[0013] In some embodiments, the DD-CRISPR enzyme comprises a Rec2
or HD2 truncation. In some embodiments, the CRISPR enzyme is
associated with the DD by way of a fusion protein. In some
embodiments, the CRISPR enzyme is fused to the DD. In other words,
the DD may be associated with the CRISPR enzyme by fusion with said
CRISPR enzyme. In some embodiments, the enzyme may be considered to
be a modified CRISPR enzyme, wherein the CRISPR enzyme is fused to
at least one destabilization domain (DD). In some embodiments, the
DD may be associated to the CRISPR enzyme via a connector protein,
for example using a system such as a marker system such as the
streptavidin-biotin system. As such, provided is a fusion of a
CRISPR enzyme with a connector protein specific for a high affinity
ligand for that connector, whereas the DD is bound to said high
affinity ligand. For example, streptavidin may be the connector
fused to the CRISPR enzyme, while biotin may be bound to the DD.
Upon co-localization, the streptavidin will bind to the biotin,
thus connecting the CRISPR enzyme to the DD. For simplicity, a
fusion of the CRISPR enzyme and the DD is preferred in some
embodiments. In some embodiments, the fusion may be to the
N-terminal end of the CRISPR enzyme. In some embodiments, at least
one DD is fused to the N-terminus of the CRISPR enzyme. In some
embodiments, the fusion may be to the C-terminal end of the CRISPR
enzyme. In some embodiments, at least one DD is fused to the
C-terminus of the CRISPR enzyme. In some embodiments, one DD may be
fused to the N-terminal end of the CRISPR enzyme with another DD
fused to the C-terminal of the CRISPR enzyme. In some embodiments,
the CRISPR enzyme is associated with at least two DDs and wherein a
first DD is fused to the N-terminus of the CRISPR enzyme and a
second DD is fused to the C-terminus of the CRISPR enzyme, the
first and second DDs being the same or different. In some
embodiments, the fusion may be to the N-terminal end of the DD. In
some embodiments, the fusion may be to the C-terminal end of the
DD. In some embodiments, the fusion may between the C-terminal end
of the CRISPR enzyme and the N-terminal end of the DD. In some
embodiments, the fusion may between the C-terminal end of the DD
and N-terminal end of the CRISPR enzyme. Less background was
observed with a DD comprising at least one N-terminal fusion than a
DD comprising at least one C terminal fusion. Combining N- and
C-terminal fusions had the least background but lowest overall
activity. Advantageously a DD is provided through at least one
N-terminal fusion or at least one N terminal fusion plus at least
one C-terminal fusion. And of course, a DD can be provided by at
least one C-terminal fusion.
[0014] In some embodiments, the DD is ER50. A corresponding
stabilizing ligand for this DD is, in some embodiments, 4HT. As
such, in some embodiments, one of the at least one DDs is ER50 and
a stabilizing ligand therefor is 4HT or CMP8 In some embodiments,
the DD is DHFR50. A corresponding stabilizing ligand for this DD
is, in some embodiments, TMP. As such, in some embodiments, one of
the at least one DDs is DHFR50 and a stabilizing ligand therefor is
TMP. In some embodiments, the DD is ER50. A corresponding
stabilizing ligand for this DD is, in some embodiments, CMP8. CMP8
may therefore be an alternative stabilizing ligand to 4HT in the
ER50 system. While it may be possible that CMP8 and 4HT can/should
be used in a competitive matter, some cell types may be more
susceptible to one or the other of these two ligands, and from this
disclosure and the knowledge in the art the skilled person can use
CMP8 and/or 4HT.
[0015] In some embodiments, one or two DDs may be fused to the
N-terminal end of the CRISPR enzyme with one or two DDs fused to
the C-terminal of the CRISPR enzyme. In some embodiments, the at
least two DDs are associated with the CRISPR enzyme and the DDs are
the same DD, i.e. the DDs are homologous. Thus, both (or two or
more) of the DDs could be ER50 DDs. This is preferred in some
embodiments. Alternatively, both (or two or more) of the DDs could
be DHFR50 DDs. This is also preferred in some embodiments. In some
embodiments, the at least two DDs are associated with the CRISPR
enzyme and the DDs are different DDs, i.e. the DDs are
heterologous. Thus, one of the DDS could be ER50 while one or more
of the or any other DDs could be DHFR50. Having two or more DDs
which are heterologous may be advantageous as it would provide a
greater level of degradation control. A tandem fusion of more than
one DD at the N or C-term may enhance degradation; and such a
tandem fusion can be, for example ER50-ER50-Cas9 or DHFR-DHFR-Ca9
It is envisaged that high levels of degradation would occur in the
absence of either stabilizing ligand, intermediate levels of
degradation would occur in the absence of one stabilizing ligand
and the presence of the other (or another) stabilizing ligand,
while low levels of degradation would occur in the presence of both
(or two of more) of the stabilizing ligands. Control may also be
imparted by having an N-terminal ER50 DD and a C-terminal DHFR50
DD.
[0016] In some embodiments, the fusion of the CRISPR enzyme with
the DD comprises a linker between the DD and the CRISPR enzyme. In
some embodiments, the linker is a GlySer linker. In some
embodiments, the DD-CRISPR enzyme further comprises at least one
Nuclear Export Signal (NES). In some embodiments, the DD-CRISPR
enzyme comprises two or more NESs. In some embodiments, the
DD-CRISPR enzyme comprises at least one Nuclear Localization Signal
(NLS). This may be in addition to an NES. In some embodiments, the
CRISPR enzyme comprises or consists essentially of or consists of a
localization (nuclear import or export) signal as, or as part of,
the linker between the CRISPR enzyme and the DD. HA or Flag tags
are also within the ambit of the invention as linkers. Applicants
use NLS and/or NES as linker and also use Glycine Serine linkers as
short as GS up to (GGGGS).sub.3 (SEQ ID NO: 27). As shown in the
Examples, more than one linker may be used and these may frame a DD
on either side (i.e. both N' an C' terminal ends).
[0017] In an aspect, the present invention provides a
polynucleotide encoding the CRISPR enzyme and associated DD. In
some embodiments, the encoded CRISPR enzyme and associated DD are
operably linked to a first regulatory element. In some embodiments,
a DD is also encoded and is operably linked to a second regulatory
element. Advantageously, the DD here is to "mop up" the stabilizing
ligand and so it is advantageously the same DD (i.e. the same type
of Domain) as that associated with the enzyme, e.g., as herein
discussed (with it understood that the term "mop up" is meant as
discussed herein and may also convey performing so as to contribute
or conclude activity). In some embodiments, the first regulatory
element is a promoter and may optionally include an enhancer. In
some embodiments, the second regulatory element is a promoter and
may optionally include an enhancer. In some embodiments, the first
regulatory element is an early promoter. In some embodiments, the
second regulatory element is a late promoter. In some embodiments,
the second regulatory element is or comprises or consists
essentially of an inducible control element, optionally the tet
system, or a repressible control element, optionally the tetr
system. An inducible promoter may be favorable e.g. rTTA to induce
tet in the presence of doxycycline.
[0018] In an aspect, the present invention provides a means for
delivering the DD-CRISPR-Cas9 complex of the invention or
polynucleotides discussed herein, e.g., particle(s) delivering
component(s) of the complex, vector(s) comprising the
polynucleotide(s) discussed herein (e.g., encoding the CRISPR
enzyme, the DD; providing RNA of the CRISPR-Cas9 complex). In some
embodiments, the vector may be a plasmid or a viral vector such as
AAV, or lentivirus. Transient transfection with plasmids, e.g.,
into HEK cells may be advantageous, especially given the size
limitations of AAV and that while SpCas9 fits into AAV, one may
reach an upper limit with additional coding as to the association
with the DD(s).
[0019] Also provided is a model that constitutively expresses the
CRISPR enzyme and associated DD. The organism may be a transgenic
and may have been transfected the present vectors or may be the
offspring of an organism so transfected. In a further aspect, the
present invention provides compositions comprising the CRISPR
enzyme and associated DD or the polynucleotides or vectors
described herein. Also provided are CRISPR-Cas9 systems comprising
guide RNAs.
[0020] Also provided is a method of treating a subject, e.g., a
subject in need thereof, comprising inducing gene editing by
transforming the subject with the polynucleotide encoding the
system or any of the present vectors and administering stabilizing
ligand to the subject. A suitable repair template may also be
provided, for example delivered by a vector comprising said repair
template. Also provided is a method of treating a subject, e.g., a
subject in need thereof, comprising inducing transcriptional
activation or repression by transforming the subject with the
polynucleotide encoding the present system or any of the present
vectors, wherein said polynucleotide or vector encodes or comprises
the catalytically inactive CRISPR enzyme and one or more associated
functional domains; the method further comprising administering a
stabilizing ligand to the subject. These methods may also include
delivering and/or expressing excess DD to the subject. Where any
treatment is occurring ex vivo, for example in a cell culture, then
it will be appreciated that the term `subject` may be replaced by
the phrase "cell or cell culture."
[0021] Compositions comprising the present system for use in said
method of treatment are also provided. A separate composition may
comprise the stabilizing ligand. A kit of parts may be provided
including such compositions. Use of the present system in the
manufacture of a medicament for such methods of treatment are also
provided. Use of the present system in screening is also provided
by the present invention, e.g., gain of function screens. Cells
which are artificially forced to overexpress a gene are be able to
down regulate the gene over time (re-establishing equilibrium) e.g.
by negative feedback loops. By the time the screen starts the
unregulated gene might be reduced again. Using an inducible Cas9
activator allows one to induce transcription right before the
screen and therefore minimizes the chance of false negative hits.
Accordingly, by use of the instant invention in screening, e.g.,
gain of function screens, the chance of false negative results may
be minimized.
[0022] In one aspect, the invention provides an engineered,
non-naturally occurring CRISPR-Cas9 system comprising a DD-Cas9
protein and a guide RNA that targets a DNA molecule encoding a gene
product in a cell, whereby the guide RNA targets the DNA molecule
encoding the gene product and the Cas9 protein cleaves the DNA
molecule encoding the gene product, whereby expression of the gene
product is altered; and, wherein the Cas9 protein and the guide RNA
do not naturally occur together. The invention comprehends the
guide RNA comprising a guide sequence fused to a tracr sequence. In
an embodiment of the invention the Cas9 protein is a type II
CRISPR-Cas9 protein and is a Cas9 protein. The invention further
comprehends coding for the Cas9 protein being codon optimized for
expression in a eukaryotic cell. In a preferred embodiment the
eukaryotic cell is a mammalian cell and in a more preferred
embodiment the mammalian cell is a human cell. In a further
embodiment of the invention, the expression of the gene product is
decreased.
[0023] In another aspect, the invention provides an engineered,
non-naturally occurring vector system comprising one or more
vectors comprising a first regulatory element operably linked to a
CRISPR-Cas9 system guide RNA that targets a DNA molecule encoding a
gene product and a second regulatory element operably linked coding
for a DD-Cas protein. Components (a) and (b) may be located on same
or different vectors of the system. The guide RNA targets the DNA
molecule encoding the gene product in a cell and the DD-Cas9
protein may cleaves the DNA molecule encoding the gene product (it
may cleave one or both strands or have substantially no nuclease
activity), whereby expression of the gene product is altered; and,
wherein the DD-Cas9 protein and the guide RNA do not naturally
occur together. The invention comprehends the guide RNA comprising
a guide sequence fused to a tracr sequence. In an embodiment of the
invention the DD-Cas9 protein is a type II DD-CRISPR-Cas9 protein
and is a DD-Cas9 protein. The invention further comprehends coding
for the DD-Cas9 protein being codon optimized for expression in a
eukaryotic cell. In a preferred embodiment the eukaryotic cell is a
mammalian cell and in a more preferred embodiment the mammalian
cell is a human cell. In a further embodiment of the invention, the
expression of the gene product is decreased.
[0024] In one aspect, the invention provides a vector system
comprising one or more vectors. In some embodiments, the system
comprises: (a) a first regulatory element operably linked to a
tracr mate sequence and one or more insertion sites for inserting
one or more guide sequences upstream of the tracr mate sequence,
wherein when expressed, the guide sequence directs
sequence-specific binding of a DD-CRISPR complex to a target
sequence in a eukaryotic cell, wherein the CRISPR complex comprises
a DD-CRISPR enzyme complexed with (1) the guide sequence that is
hybridized to the target sequence, and (2) the tracr mate sequence
that is hybridized to the tracr sequence; and (b) a second
regulatory element operably linked to an enzyme-coding sequence
encoding said DD-CRISPR enzyme comprising at least one nuclear
localization sequence and/or at least one NES; wherein components
(a) and (b) are located on the same or different vectors of the
system. In some embodiments, component (a) further comprises the
tracr sequence downstream of the tracr mate sequence under the
control of the first regulatory element. In some embodiments,
component (a) further comprises two or more guide sequences
operably linked to the first regulatory element, wherein when
expressed, each of the two or more guide sequences direct sequence
specific binding of a DD-CRISPR complex to a different target
sequence in a eukaryotic cell. In some embodiments, the system
comprises the tracr sequence under the control of a third
regulatory element, such as a polymerase III promoter. In some
embodiments, the tracr sequence exhibits at least 50%, 60%, 70%,
80%, 90%, 95%, or 99% of sequence complementarity along the length
of the tracr mate sequence when optimally aligned. Determining
optimal alignment is within the purview of one of skill in the art.
For example, there are publically and commercially available
alignment algorithms and programs such as, but not limited to,
ClustalW, Smith-Waterman in matlab, Bowtie, Geneious, Biopython and
SeqMan. In some embodiments, the DD-CRISPR complex comprises one or
more nuclear localization sequences and/or one or more NES of
sufficient strength to drive accumulation of said CRISPR complex in
a detectable amount in or out of the nucleus of a eukaryotic cell.
Without wishing to be bound by theory, it is believed that a
nuclear localization sequence and/or NES is not necessary for
DD-CRISPR complex activity in eukaryotes, but that including such
sequences enhances activity of the system, especially as to
targeting nucleic acid molecules in the nucleus and/or having
molecules exit the nucleus. In some embodiments, the DD-CRISPR
enzyme is a type II DD-CRISPR system enzyme andis a DD-Cas9 enzyme.
In some embodiments, the DD-Cas9 enzyme is derived from S.
pneumoniae, S. pyogenes, S. thermophiles, F. novicida or S. aureus
Cas9 (e.g., a Cas9 of one of these organisms modified to have or be
associated with at least one DD), and may include further mutations
or alterations or be a chimeric Cas9. The enzyme may be a DD-Cas9
homolog or ortholog. In some embodiments, the DD-CRISPR enzyme is
codon-optimized for expression in a eukaryotic cell. In some
embodiments, the DD-CRISPR enzyme directs cleavage of one or two
strands at the location of the target sequence. In some
embodiments, the DD-CRISPR enzyme lacks DNA strand cleavage
activity. In some embodiments, the first regulatory element is a
polymerase III promoter. In some embodiments, the second regulatory
element is a polymerase II promoter. In some embodiments, the guide
sequence is at least 15, 16, 17, 18, 19, 20, 25 nucleotides, or
between 10-30, or between 15-25, or between 15-20 nucleotides in
length. In general, and throughout this specification, the term
"vector" refers to a nucleic acid molecule capable of transporting
another nucleic acid to which it has been linked. Vectors include,
but are not limited to, nucleic acid molecules that are
single-stranded, double-stranded, or partially double-stranded;
nucleic acid molecules that comprise one or more free ends, no free
ends (e.g., circular); nucleic acid molecules that comprise DNA,
RNA, or both; and other varieties of polynucleotides known in the
art. One type of vector is a "plasmid," which refers to a circular
double stranded DNA loop into which additional DNA segments can be
inserted, such as by standard molecular cloning techniques. Another
type of vector is a viral vector, wherein virally-derived DNA or
RNA sequences are present in the vector for packaging into a virus
(e.g., retroviruses, replication defective retroviruses,
adenoviruses, replication defective adenoviruses, and
adeno-associated viruses). Viral vectors also include
polynucleotides carried by a virus for transfection into a host
cell. Certain vectors are capable of autonomous replication in a
host cell into which they are introduced (e.g., bacterial vectors
having a bacterial origin of replication and episomal mammalian
vectors). Other vectors (e.g., non-episomal mammalian vectors) are
integrated into the genome of a host cell upon introduction into
the host cell, and thereby are replicated along with the host
genome. Moreover, certain vectors are capable of directing the
expression of genes to which they are operatively-linked. Such
vectors are referred to herein as "expression vectors." Common
expression vectors of utility in recombinant DNA techniques are
often in the form of plasmids.
[0025] Recombinant expression vectors can comprise a nucleic acid
of the invention in a form suitable for expression of the nucleic
acid in a host cell, which means that the recombinant expression
vectors include one or more regulatory elements, which may be
selected on the basis of the host cells to be used for expression,
that is operatively-linked to the nucleic acid sequence to be
expressed. Within a recombinant expression vector, "operably
linked" is intended to mean that the nucleotide sequence of
interest is linked to the regulatory element(s) in a manner that
allows for expression of the nucleotide sequence (e.g., in an in
vitro transcription/translation system or in a host cell when the
vector is introduced into the host cell).
[0026] The term "regulatory element" is intended to include
promoters, enhancers, internal ribosomal entry sites (IRES), and
other expression control elements (e.g., transcription termination
signals, such as polyadenylation signals and poly-U sequences).
Such regulatory elements are described, for example, in Goeddel,
GENE EXPRESSION TECHNOLOGY: METHODS IN ENZYMOLOGY 185, Academic
Press, San Diego, Calif. (1990). Regulatory elements include those
that direct constitutive expression of a nucleotide sequence in
many types of host cell and those that direct expression of the
nucleotide sequence only in certain host cells (e.g.,
tissue-specific regulatory sequences). A tissue-specific promoter
may direct expression primarily in a desired tissue of interest,
such as muscle, neuron, bone, skin, blood, specific organs (e.g.,
liver, pancreas), or particular cell types (e.g., lymphocytes).
Regulatory elements may also direct expression in a
temporal-dependent manner, such as in a cell-cycle dependent or
developmental stage-dependent manner, which may or may not also be
tissue or cell-type specific. In some embodiments, a vector
comprises one or more pol III promoter (e.g., 1, 2, 3, 4, 5, or
more pol III promoters), one or more pol II promoters (e.g., 1, 2,
3, 4, 5, or more pol II promoters), one or more pol I promoters
(e.g., 1, 2, 3, 4, 5, or more pol I promoters), or combinations
thereof. Examples of pol III promoters include, but are not limited
to, U6 and H1 promoters. Examples of pol II promoters include, but
are not limited to, the retroviral Rous sarcoma virus (RSV) LTR
promoter (optionally with the RSV enhancer), the cytomegalovirus
(CMV) promoter (optionally with the CMV enhancer) [see, e.g.,
Boshart et al, Cell, 41:521-530 (1985)], the SV40 promoter, the
dihydrofolate reductase promoter, the .beta.-actin promoter, the
phosphoglycerol kinase (PGK) promoter, and the EF1.alpha. promoter.
Also encompassed by the term "regulatory element" are enhancer
elements, such as WPRE; CMV enhancers; the R-U5' segment in LTR of
HTLV-I (Mol. Cell. Biol., Vol. 8(1), p. 466-472, 1988); SV40
enhancer; and the intron sequence between exons 2 and 3 of rabbit
.beta.-globin (Proc. Natl. Acad. Sci. USA., Vol. 78(3), p. 1527-31,
1981). It will be appreciated by those skilled in the art that the
design of the expression vector can depend on such factors as the
choice of the host cell to be transformed, the level of expression
desired, etc. A vector can be introduced into host cells to thereby
produce transcripts, proteins, or peptides, including fusion
proteins or peptides, encoded by nucleic acids as described herein
(e.g., clustered regularly interspersed short palindromic repeats
(CRISPR) transcripts, proteins, enzymes, mutant forms thereof,
fusion proteins thereof, etc.).
[0027] Advantageous vectors include lentiviruses and
adeno-associated viruses, and types of such vectors can also be
selected for targeting particular types of cells.
[0028] In one aspect, the invention provides a vector comprising a
regulatory element operably linked to an enzyme-coding sequence
encoding a DD-CRISPR enzyme comprising one or more nuclear
localization sequences and/or NES. In some embodiments, said
regulatory element drives transcription of the DD-CRISPR enzyme in
a eukaryotic cell such that said DD-CRISPR enzyme accumulates in a
detectable amount in the nucleus of the eukaryotic cell and/or is
exported from the nucleus. In some embodiments, the regulatory
element is a polymerase II promoter. In some embodiments, the
DD-CRISPR enzyme is a type II DD-CRISPR system enzyme and is a
DD-Cas9 enzyme. In some embodiments, the DD-Cas9 enzyme is derived
from S. pneumoniae, S. pyogenes, S. thermophilus, F. novicida or S.
aureus Cas9 (e.g., modified to have or be associated with at least
one DD), and may include further alteration or mutation of the
Cas9, and can be a chimeric Cas9. In some embodiments, the
DD-CRISPR enzyme is codon-optimized for expression in a eukaryotic
cell. In some embodiments, the DD-CRISPR enzyme directs cleavage of
one or two strands at the location of the target sequence. In some
embodiments, the DD-CRISPR enzyme lacks or substantially DNA strand
cleavage activity (e.g., no more than 5% nuclease activity as
compared with a wild type enzyme or enzyme not having the mutation
or alteration that decreases nuclease activity).
[0029] In one aspect, the invention provides a DD-CRISPR enzyme
comprising one or more nuclear localization sequences and/or NES of
sufficient strength to drive accumulation of said DD-CRISPR enzyme
in a detectable amount in and/or out of the nucleus of a eukaryotic
cell. In some embodiments, the DD-CRISPR enzyme is a type II
DD-CRISPR system enzyme and is a DD-Cas9 enzyme. In some
embodiments, the DD-Cas9 enzyme is derived from S. pneumoniae, S.
pyogenes, S. thermophilus, F. novicida or S. aureus Cas9 (e.g.,
modified to have or be associated with at least one DD), and may
include further alteration or mutation of the Cas9, and can be a
chimeric Cas9. In some embodiments, the DD-CRISPR enzyme is
codon-optimized for expression in a eukaryotic cell. In some
embodiments, the DD-CRISPR enzyme directs cleavage of one or two
strands at the location of the target sequence. In some
embodiments, the DD-CRISPR enzyme lacks or substantially DNA strand
cleavage activity (e.g., no more than 5% nuclease activity as
compared with a wild type enzyme or enzyme not having the mutation
or alteration that decreases nuclease activity).
[0030] In one aspect, the invention provides a eukaryotic host cell
comprising (a) a first regulatory element operably linked to a
tracr mate sequence and one or more insertion sites for inserting
one or more guide sequences upstream of the tracr mate sequence,
wherein when expressed, the guide sequence directs
sequence-specific binding of a DD-CRISPR complex to a target
sequence in a eukaryotic cell, wherein the DD-CRISPR complex
comprises a DD-CRISPR enzyme complexed with (1) the guide sequence
that is hybridized to the target sequence, and (2) the tracr mate
sequence that is hybridized to the tracr sequence; and/or (b) a
second regulatory element operably linked to an enzyme-coding
sequence encoding said DD-CRISPR enzyme comprising at least one
nuclear localization sequence and/or NES. In some embodiments, the
host cell comprises components (a) and (b). In some embodiments,
component (a), component (b), or components (a) and (b) are stably
integrated into a genome of the host eukaryotic cell. In some
embodiments, component (a) further comprises the tracr sequence
downstream of the tracr mate sequence under the control of the
first regulatory element. In some embodiments, component (a)
further comprises two or more guide sequences operably linked to
the first regulatory element, wherein when expressed, each of the
two or more guide sequences direct sequence specific binding of a
DD-CRISPR complex to a different target sequence in a eukaryotic
cell. In some embodiments, the eukaryotic host cell further
comprises a third regulatory element, such as a polymerase III
promoter, operably linked to said tracr sequence. In some
embodiments, the tracr sequence exhibits at least 50%, 60%, 70%,
80%, 90%, 95%, or 99% of sequence complementarity along the length
of the tracr mate sequence when optimally aligned. In some
embodiments, the DD-CRISPR enzyme comprises one or more nuclear
localization sequences and/or nuclear export sequences or NES of
sufficient strength to drive accumulation of said CRISPR enzyme in
a detectable amount in and/or out of the nucleus of a eukaryotic
cell. In some embodiments, the DD-CRISPR enzyme is a type II CRISPR
system enzyme and is a Cas9 enzyme. In some embodiments, the
DD-Cas9 enzyme is derived from S. pneumoniae, S. pyogenes, S.
thermophdus, F. novicida or S. aureus Cas9 (e.g., modified to have
or be associated with at least one DD), and may include further
alteration or mutation of the Cas9, and can be a chimeric Cas9. In
some embodiments, the DD-CRISPR enzyme is codon-optimized for
expression in a eukaryotic cell. In some embodiments, the DD-CRISPR
enzyme directs cleavage of one or two strands at the location of
the target sequence. In some embodiments, the DD-CRISPR enzyme
lacks or substantially DNA strand cleavage activity (e.g., no more
than 5% nuclease activity as compared with a wild type enzyme or
enzyme not having the mutation or alteration that decreases
nuclease activity). In some embodiments, the first regulatory
element is a polymerase III promoter. In some embodiments, the
second regulatory element is a polymerase II promoter. In some
embodiments, the guide sequence is at least 15, 16, 17, 18, 19, 20,
25 nucleotides, or between 10-30, or between 15-25, or between
15-20 nucleotides in length. In an aspect, the invention provides a
non-human eukaryotic organism; preferably a multicellular
eukaryotic organism, comprising a eukaryotic host cell according to
any of the described embodiments. In other aspects, the invention
provides a eukaryotic organism; preferably a multicellular
eukaryotic organism, comprising a eukaryotic host cell according to
any of the described embodiments. The organism in some embodiments
of these aspects may be an animal; for example a mammal such as a
mouse. Also, the organism may be an arthropod such as an insect,
for instance, a fly (especially fruit flies including model
organisms such as Drosophila melanogaster as well as agricultural
pests such as olive fly) or a mosquito. Indeed, insect and
arthropod models, disease vectors and pests are preferred,
including moths, mosquitoes, boring insects, fruit flies etc. The
organism may be a nematode such as C. elegans. The organism also
may be a plant. Further, the organism may be a fungus.
[0031] With respect to use of the CRISPR-Cas system generally,
mention is made of the documents, including patent applications,
patents, and patent publications cited throughout this disclosure
as embodiments of the invention can be used as in those documents.
CRISPR-Cas system(s) (e.g., single or multiplexed) can be used in
conjunction with recent advances in crop genomics. Such CRISPR-Cas
system(s) can be used to perform efficient and cost effective plant
gene or genome interrogation or editing or manipulation--for
instance, for rapid investigation and/or selection and/or
interrogations and/or comparison and/or manipulations and/or
transformation of plant genes or genomes; e.g., to create,
identify, develop, optimize, or confer trait(s) or
characteristic(s) to plant(s) or to transform a plant genome. There
can accordingly be improved production of plants, new plants with
new combinations of traits or characteristics or new plants with
enhanced traits. Such CRISPR-Cas system(s) can be used with regard
to plants in Site-Directed Integration (SDI) or Gene Editing (GE)
or any Near Reverse Breeding (NRB) or Reverse Breeding (RB)
techniques. With respect to use of the CRISPR-Cas system in plants,
mention is made of the University of Arizona website "CRISPR-PLANT"
(http://www.genome.arizona.edu/crispr/) (supported by Penn State
and AGI). Embodiments of the invention can be used in genome
editing in plants or where RNAi or similar genome editing
techniques have been used previously; see, e.g., Nekrasov, "Plant
genome editing made easy: targeted mutagenesis in model and crop
plants using the CRISPR/Cas system," Plant Methods 2013, 9:39
(doi:10.1186/1746-4811-9-39); Brooks, "Efficient gene editing in
tomato in the first generation using the CRISPR/Cas9 system," Plant
Physiology September 2014 pp 114.247577; Shan, "Targeted genome
modification of crop plants using a CRISPR-Cas system," Nature
Biotechnology 31, 686-688 (2013); Feng, "Efficient genome editing
in plants using a CRISPR/Cas system," Cell Research (2013)
23:1229-1232. doi:10.1038/cr.2013.114; published online 20 Aug.
2013; Xie, "RNA-guided genome editing in plants using a CRISPR-Cas
system," Mol Plant. 2013 November; 6(6):1975-83. doi:
10.1093/mp/sst119. Epub 2013 Aug. 17; Xu, "Gene targeting using the
Agrobacterium tumefaciens-mediated CRISPR-Cas system in rice," Rice
2014, 7:5 (2014), Zhou et al., "Exploiting SNPs for biallelic
CRISPR mutations in the outcrossing woody perennial Populus reveals
4-coumarate: CoA ligase specificity and Redundancy," New
Phytologist (2015) (Forum) 1-4 (available online only at
www.newphytologist.com); Caliando et al, "Targeted DNA degradation
using a CRISPR device stably carried in the host genome, NATURE
COMMUNICATIONS 6:6989, DOI: 10.1038/ncomms7989,
www.nature.com/naturecommunications DOI: 10.1038/ncomms7989; U.S.
Pat. No. 6,603,061--Agrobacterium-Mediated Plant Transformation
Method; U.S. Pat. No. 7,868,149--Plant Genome Sequences and Uses
Thereof and US 2009/0100536--Transgenic Plants with Enhanced
Agronomic Traits, all the contents and disclosure of each of which
are herein incorporated by reference in their entirety. In the
practice of the invention, the contents and disclosure of Morrell
et al "Crop genomics: advances and applications," Nat Rev Genet.
2011 Dec. 29; 13(2):85-96; each of which is incorporated by
reference herein including as to how herein embodiments may be used
as to plants. Accordingly, reference herein to animal cells may
also apply, mutatis mutandis, to plant cells unless otherwise
apparent.
[0032] In one aspect, the invention provides a kit comprising one
or more of the components described herein. In some embodiments,
the kit comprises a vector system and instructions for using the
kit. In some embodiments, the vector system comprises (a) a first
regulatory element operably linked to a tracr mate sequence and one
or more insertion sites for inserting one or more guide sequences
upstream of the tracr mate sequence, wherein when expressed, the
guide sequence directs sequence-specific binding of a CRISPR
complex to a target sequence in a eukaryotic cell, wherein the
CRISPR complex comprises a CRISPR enzyme complexed with (1) the
guide sequence that is hybridized to the target sequence, and (2)
the tracr mate sequence that is hybridized to the tracr sequence;
and/or (b) a second regulatory element operably linked to an
enzyme-coding sequence encoding said CRISPR enzyme comprising a
nuclear localization sequence. In some embodiments, the kit
comprises components (a) and (b) located on the same or different
vectors of the system. In some embodiments, component (a) further
comprises the tracr sequence downstream of the tracr mate sequence
under the control of the first regulatory element. In some
embodiments, component (a) further comprises two or more guide
sequences operably linked to the first regulatory element, wherein
when expressed, each of the two or more guide sequences direct
sequence specific binding of a CRISPR complex to a different target
sequence in a eukaryotic cell. In some embodiments, the system
further comprises a third regulatory element, such as a polymerase
III promoter, operably linked to said tracr sequence. In some
embodiments, the tracr sequence exhibits at least 50%, 60%, 70%,
80%, 90%, 95%, or 99% of sequence complementarity along the length
of the tracr mate sequence when optimally aligned. In some
embodiments, the CRISPR enzyme comprises one or more nuclear
localization sequences of sufficient strength to drive accumulation
of said CRISPR enzyme in a detectable amount in the nucleus of a
eukaryotic cell. The CRISPR enzyme is a type II CRISPR system
enzyme and is a Cas9 enzyme. In some embodiments, the Cas9 enzyme
is derived from S. pneumoniae, S. pyogenes, S. thermophdus, F.
novicida or S. aureus Cas9 (e.g., modified to have or be associated
with at least one DD), and may include further alteration or
mutation of the Cas9, and can be a chimeric Cas9. In some
embodiments, the DD-CRISPR enzyme is codon-optimized for expression
in a eukaryotic cell. In some embodiments, the DD-CRISPR enzyme
directs cleavage of one or two strands at the location of the
target sequence. In some embodiments, the DD-CRISPR enzyme lacks or
substantially DNA strand cleavage activity (e.g., no more than 5%
nuclease activity as compared with a wild type enzyme or enzyme not
having the mutation or alteration that decreases nuclease
activity). In some embodiments, the first regulatory element is a
polymerase III promoter. In some embodiments, the second regulatory
element is a polymerase II promoter. In some embodiments, the guide
sequence is at least 15, 16, 17, 18, 19, 20, 25 nucleotides, or
between 10-30, or between 15-25, or between 15-20 nucleotides in
length.
[0033] In one aspect, the invention provides a method of modifying
a target polynucleotide in a eukaryotic cell. In some embodiments,
the method comprises allowing a DD-CRISPR complex to bind to the
target polynucleotide, e.g., to effect cleavage of said target
polynucleotide, thereby modifying the target polynucleotide,
wherein the DD-CRISPR complex comprises a DD-CRISPR enzyme
complexed with a guide sequence hybridized to a target sequence
within said target polynucleotide, wherein said guide sequence is
linked to a tracr mate sequence which in turn hybridizes to a tracr
sequence. In some embodiments, said cleavage comprises cleaving one
or two strands at the location of the target sequence by said
DD-CRISPR enzyme. In some embodiments, said cleavage results in
decreased transcription of a target gene. In some embodiments, the
method further comprises repairing said cleaved target
polynucleotide by homologous recombination with an exogenous
template polynucleotide, wherein said repair results in a mutation
comprising an insertion, deletion, or substitution of one or more
nucleotides of said target polynucleotide. In some embodiments,
said mutation results in one or more amino acid changes in a
protein expressed from a gene comprising the target sequence. In
some embodiments, the method further comprises delivering one or
more vectors to said eukaryotic cell, wherein the one or more
vectors drive expression of one or more of: the DD-CRISPR enzyme,
the guide sequence linked to the tracr mate sequence, and the tracr
sequence. In some embodiments, said vectors are delivered to the
eukaryotic cell in a subject. In some embodiments, said modifying
takes place in said eukaryotic cell in a cell culture. In some
embodiments, the method further comprises isolating said eukaryotic
cell from a subject prior to said modifying. In some embodiments,
the method further comprises returning said eukaryotic cell and/or
cells derived therefrom to said subject.
[0034] In one aspect, the invention provides a method of modifying
expression of a polynucleotide in a eukaryotic cell. In some
embodiments, the method comprises allowing a DD-CRISPR complex to
bind to the polynucleotide such that said binding results in
increased or decreased expression of said polynucleotide; wherein
the DD-CRISPR complex comprises a DD-CRISPR enzyme complexed with a
guide sequence hybridized to a target sequence within said
polynucleotide, wherein said guide sequence is linked to a tracr
mate sequence which in turn hybridizes to a tracr sequence. In some
embodiments, the method further comprises delivering one or more
vectors to said eukaryotic cells, wherein the one or more vectors
drive expression of one or more of: the DD-CRISPR enzyme, the guide
sequence linked to the tracr mate sequence, and the tracr
sequence.
[0035] In one aspect, the invention provides a method of generating
a model eukaryotic cell comprising a mutated disease gene. In some
embodiments, a disease gene is any gene associated an increase in
the risk of having or developing a disease. In some embodiments,
the method comprises (a) introducing one or more vectors into a
eukaryotic cell, wherein the one or more vectors drive expression
of one or more of: a DD-CRISPR enzyme, a guide sequence linked to a
tracr mate sequence, and a tracr sequence; and (b) allowing a
DD-CRISPR complex to bind to a target polynucleotide, e.g., to
effect cleavage of the target polynucleotide within said disease
gene, wherein the DD-CRISPR complex comprises the DD-CRISPR enzyme
complexed with (1) the guide sequence that is hybridized to the
target sequence within the target polynucleotide, and (2) the tracr
mate sequence that is hybridized to the tracr sequence, thereby
generating a model eukaryotic cell comprising a mutated disease
gene. In some embodiments, said cleavage comprises cleaving one or
two strands at the location of the target sequence by said
DD-CRISPR enzyme. In some embodiments, said cleavage results in
decreased transcription of a target gene. In some embodiments, the
method further comprises repairing said cleaved target
polynucleotide by homologous recombination with an exogenous
template polynucleotide, wherein said repair results in a mutation
comprising an insertion, deletion, or substitution of one or more
nucleotides of said target polynucleotide. In some embodiments,
said mutation results in one or more amino acid changes in a
protein expression from a gene comprising the target sequence.
[0036] In one aspect, the invention provides a method for
developing a biologically active agent that modulates a cell
signaling event associated with a disease gene. In some
embodiments, a disease gene is any gene associated an increase in
the risk of having or developing a disease. In some embodiments,
the method comprises (a) contacting a test compound with a model
cell of any one of the described embodiments; and (b) detecting a
change in a readout that is indicative of a reduction or an
augmentation of a cell signaling event associated with said
mutation in said disease gene, thereby developing said biologically
active agent that modulates said cell signaling event associated
with said disease gene.
[0037] In one aspect, the invention provides a recombinant
polynucleotide comprising a guide sequence upstream of a tracr mate
sequence, wherein the guide sequence when expressed directs
sequence-specific binding of a DD-CRISPR complex to a corresponding
target sequence present in a eukaryotic cell. In some embodiments,
the target sequence is a viral sequence present in a eukaryotic
cell. In some embodiments, the target sequence is a proto-oncogene
or an oncogene.
[0038] In one aspect the invention provides for a method of
selecting one or more cell(s) by introducing one or more mutations
in a gene in the one or more cell (s), the method comprising:
introducing one or more vectors into the cell (s), wherein the one
or more vectors drive expression of one or more of: a DD-CRISPR
enzyme, a guide sequence linked to a tracr mate sequence, a tracr
sequence, and an editing template; wherein the editing template
comprises the one or more mutations that abolish DD-CRISPR enzyme
cleavage; allowing homologous recombination of the editing template
with the target polynucleotide in the cell(s) to be selected;
allowing a CRISPR complex to bind to a target polynucleotide to
effect cleavage of the target polynucleotide within said gene,
wherein the DD-CRISPR complex comprises the DD-CRISPR enzyme
complexed with (1) the guide sequence that is hybridized to the
target sequence within the target polynucleotide, and (2) the tracr
mate sequence that is hybridized to the tracr sequence, wherein
binding of the DD-CRISPR complex to the target polynucleotide
induces cell death, thereby allowing one or more cell(s) in which
one or more mutations have been introduced to be selected. The
DD-CRISPR enzyme is DD-Cas9. In another aspect of the invention the
cell to be selected may be a eukaryotic cell. Aspects of the
invention allow for selection of specific cells without requiring a
selection marker or a two-step process that may include a
counter-selection system. The cell(s) may be prokaryotic or
eukaryotic cells.
[0039] With respect to mutations of the DD-CRISPR enzyme, when the
enzyme is not SpCas9, mutations may be made at any or all residues
corresponding to positions 10, 762, 840, 854, 863 and/or 986 of
SpCas9 (which may be ascertained for instance by standard sequence
comparison tools). In particular, any or all of the following
mutations are preferred in SpCas9: D10A, E762A, H840A, N854A, N863A
and/or D986A; as well as conservative substitution for any of the
replacement amino acids is also envisaged. In an aspect the
invention provides as to any or each or all embodiments
herein-discussed wherein the DD-CRISPR enzyme comprises at least
one or more, or at least two or more mutations, wherein the at
least one or more mutation or the at least two or more mutations is
as to D10, E762, H840, N854, N863, or D986 according to SpCas9
protein, e.g., D10A, E762A, H840A, N854A, N863A and/or D986A as to
SpCas9, or N580 according to SaCas9, e.g., N580A as to SaCas9, or
any corresponding mutation(s) in a Cas9 of an ortholog to Sp or Sa,
or the CRISPR enzyme comprises at least one mutation wherein at
least H840 or N863A as to Sp Cas9 or N580A as to Sa Cas9 is
mutated; e.g., wherein the CRISPR enzyme comprises H840A, or D10A
and H840A, or D10A and N863A, according to SpCas9 protein, or any
corresponding mutation(s) in a Cas9 of an ortholog to Sp protein or
Sa protein.
[0040] In a further aspect, the invention involves a
computer-assisted method for identifying or designing potential
compounds to fit within or bind to DD-CRISPR-Cas9 system or a
functional portion thereof or vice versa (a computer-assisted
method for identifying or designing potential DD-CRISPR-Cas9
systems or a functional portion thereof for binding to desired
compounds) or a computer-assisted method for identifying or
designing potential DD-CRISPR-Cas9 systems (e.g., with regard to
predicting areas of the DD-CRISPR-Cas9 system to be able to be
manipulated--for instance, based on crystal structure data or based
on data of Cas9 orthologs, or with respect to where a functional
group such as an activator or repressor can be attached to the
DD-CRISPR-Cas9 system, or as to Cas9 truncations or as to designing
nickases), said method comprising: using a computer system, e.g., a
programmed computer comprising a processor, a data storage system,
an input device, and an output device, the steps of: (a) inputting
into the programmed computer through said input device data
comprising the three-dimensional co-ordinates of a subset of the
atoms from or pertaining to the DD-CRISPR-Cas9 crystal structure,
e.g., in the DD-CRISPR-Cas9 system binding domain or alternatively
or additionally in domains that vary based on variance among Cas9
orthologs or as to Cas9s or as to nickases or as to functional
groups, optionally with structural information from CRISPR-Cas9
system complex(es), thereby generating a data set; (b) comparing,
using said processor, said data set to a computer database of
structures stored in said computer data storage system, e.g.,
structures of compounds that bind or putatively bind or that are
desired to bind to a DD-CRISPR-Cas9 system or as to DD-Cas9
orthologs (e.g., as Cas9s or as to domains or regions that vary
amongst Cas9 orthologs) or as to the DD-CRISPR-Cas9 crystal
structure or as to nickases or as to functional groups; (c)
selecting from said database, using computer methods,
structure(s)--e.g., DD-CRISPR-Cas9 structures that may bind to
desired structures, desired structures that may bind to certain
DD-CRISPR-Cas9 structures, portions of the DD-CRISPR-Cas9 system
that may be manipulated, e.g., based on data from other portions of
the DD-CRISPR-Cas9 crystal structure and/or from DD-Cas9 orthologs,
truncated Cas9s, novel nickases or particular functional groups, or
positions for attaching functional groups to or mutating
DD-CRISPR-Cas9 systems; (d) constructing, using computer methods, a
model of the selected structure(s); and (e) outputting to said
output device the selected structure(s); and optionally
synthesizing one or more of the selected structure(s); and further
optionally testing said synthesized selected structure(s) as or in
a DD-CRISPR-Cas9 system; or, said method comprising: providing the
co-ordinates of at least two atoms of the DD-CRISPR-Cas9 crystal
structure, e.g., at least two atoms of the herein cited materials
or co-ordinates of at least a sub-domain of the DD-CRISPR-Cas9
crystal structure ("selected co-ordinates"), providing the
structure of a candidate comprising a binding molecule or of
portions of the DD-CRISPR-Cas9 system that may be manipulated,
e.g., based on data from other portions of the DD-CRISPR-Cas9
crystal structure and/or from Cas9 orthologs, or the structure of
functional groups, and fitting the structure of the candidate to
the selected co-ordinates, to thereby obtain product data
comprising DD-CRISPR-Cas9 structures that may bind to desired
structures, desired structures that may bind to certain
DD-CRISPR-Cas9 structures, portions of the CRISPR-Cas9 system that
may be manipulated, truncated Cas9s, novel nickases, or particular
functional groups, or positions for attaching functional groups or
for mutating DD-CRISPR-Cas9 systems, with output thereof; and
optionally synthesizing compound(s) from said product data and
further optionally comprising testing said synthesized compound(s)
as or in a DD-CRISPR-Cas9 system. The testing can comprise
analyzing the DD-CRISPR-Cas9 system resulting from said synthesized
selected structure(s), e.g., with respect to binding, or performing
a desired function. The output in the foregoing methods can
comprise data transmission, e.g., transmission of information via
telecommunication, telephone, video conference, mass communication,
e.g., presentation such as a computer presentation (e.g.
POWERPOINT), internet, email, documentary communication such as a
computer program (e.g. WORD) document and the like. Accordingly,
the invention also comprehends computer readable media containing:
atomic co-ordinate data according to the herein cited materials,
said data defining the three dimensional structure of
DD-CRISPR-Cas9 or at least one sub-domain thereof, or structure
factor data for CRISPR-Cas9, said structure factor data being
derivable from the herein cited materials. The computer readable
media can also contain any data of the foregoing methods. The
invention further comprehends methods a computer system for
generating or performing rational design as in the foregoing
methods containing either: atomic co-ordinate data according to
herein cited materials, said data defining the three dimensional
structure of DD-CRISPR-Cas9 or at least one sub-domain thereof, or
structure factor data for CRISPR-Cas9, said structure factor data
being derivable from the atomic co-ordinate data of herein cited
materials. The invention further comprehends a method of doing
business comprising providing to a user the computer system or the
media or the three dimensional structure of DD-CRISPR-Cas9 or at
least one sub-domain thereof, or structure factor data for
DD-CRISPR-Cas9, said structure set forth in and said structure
factor data being derivable from the atomic co-ordinate data of
herein cited materials, or the herein computer media or a herein
data transmission.
[0041] A "binding site" or an "active site" comprises or consists
essentially of or consists of a site (such as an atom, a functional
group of an amino acid residue or a plurality of such atoms and/or
groups) in a binding cavity or region, which may bind to a compound
such as a nucleic acid molecule, which is/are involved in binding.
By "fitting", is meant determining by automatic, or semi-automatic
means, interactions between one or more atoms of a candidate
molecule and at least one atom of a structure of the invention, and
calculating the extent to which such interactions are stable.
Interactions include attraction and repulsion, brought about by
charge, steric considerations and the like. Various computer-based
methods for fitting are described further By "root mean square (or
rms) deviation", we mean the square root of the arithmetic mean of
the squares of the deviations from the mean. By a "computer
system", is meant the hardware means, software means and data
storage means used to analyze atomic coordinate data. The minimum
hardware means of the computer-based systems of the present
invention typically comprises a central processing unit (CPU),
input means, output means and data storage means. Desirably a
display or monitor is provided to visualize structure data. The
data storage means may be RAM or means for accessing computer
readable media of the invention. Examples of such systems are
computer and tablet devices running Unix, Windows or Apple
operating systems. By "computer readable media", is meant any
medium or media, which can be read and accessed directly or
indirectly by a computer e.g., so that the media is suitable for
use in the above-mentioned computer system. Such media include, but
are not limited to: magnetic storage media such as floppy discs,
hard disc storage medium and magnetic tape; optical storage media
such as optical discs or CD-ROM; electrical storage media such as
RAM and ROM; thumb drive devices; cloud storage devices and hybrids
of these categories such as magnetic/optical storage media.
[0042] In particular embodiments of the invention, the
conformational variations in the crystal structures of the
DD-CRISPR-Cas9 system or of components of the DD-CRISPR-Cas9
provide important and critical information about the flexibility or
movement of protein structure regions relative to nucleotide (RNA
or DNA) structure regions that may be important for DD-CRISPR-Cas
system function. The structural information provided for Cas9
(e.g., S. pyogenes Cas9) in the herein cited materials may be used
to further engineer and optimize the herein DD-CRISPR-Cas system
and this may be extrapolated to interrogate structure-function
relationships in other CRISPR enzyme, e.g., DD-CRISPR enzyme
systems as well, e.g., other Type II CRISPR enzyme systems (for
instance other Type II DD-CRISPR enzyme systems). The invention
comprehends optimized functional DD-CRISPR-Cas enzyme systems. In
particular the DD-CRISPR enzyme comprises one or more mutations
that converts it to a DNA binding protein to which functional
domains exhibiting a function of interest may be recruited or
appended or inserted or attached. In certain embodiments, the
CRISPR enzyme comprises one or more mutations which include but are
not limited to D10A, E762A, H840A, N854A, N863A or D986A (based on
the amino acid position numbering of a S. pyogenes Cas9) and/or the
one or more mutations is in a RuvC1 or HNH domain of the DD-CRISPR
enzyme and/or is a mutation as otherwise as discussed herein. In
some embodiments, the DD-CRISPR enzyme has one or more mutations in
a catalytic domain, wherein when transcribed, the tracr mate
sequence hybridizes to the tracr sequence and the guide sequence
directs sequence-specific binding of a DD-CRISPR complex to the
target sequence, and wherein the enzyme further comprises a
functional domain (e.g., for providing the destabilized domain or
contributing thereto). The structural information provided in the
herein cited materials allows for interrogation of sgRNA (or
chimeric RNA) interaction with the target DNA and the CRISPR enzyme
(e.g., Cas9; for instance DD-CRISPR enzyme, e.g., DD-Cas9))
permitting engineering or alteration of sgRNA structure to optimize
functionality of the entire DD-CRISPR-Cas system. For example,
loops of the sgRNA may be extended, without colliding with the Cas9
protein by the insertion of adaptor proteins that can bind to RNA.
These adaptor proteins can further recruit effector proteins or
fusions which comprise one or more functional domains. The
functional domain may comprise, consist essentially of or consist
of a transcriptional activation domain, e.g. VP64. The functional
domain may comprise, consist essentially of a transcription
repression domain, e.g., KRAB. In some embodiments, the
transcription repression domain is or comprises or consists
essentially of SID, or concatemers of SID (e.g. SID4X). In some
embodiments, the functional domain comprise, consist essentially of
an epigenetic modifying domain, such that an epigenetic modifying
enzyme is provided. In some embodiments, the functional domain
comprise, consist essentially of an activation domain, which may be
the P65 activation domain.
[0043] Aspects of the invention encompass a non-naturally occurring
or engineered composition that may comprise a guide RNA (sgRNA)
comprising a guide sequence capable of hybridizing to a target
sequence in a genomic locus of interest in a cell and a DD-CRISPR
enzyme that may comprise at least one or more nuclear localization
sequences, wherein the DD-CRISPR enzyme comprises one or two or
more mutations, such that the enzyme has altered or diminished
nuclease activity compared with the wild type enzyme, wherein at
least one loop of the sgRNA is modified by the insertion of
distinct RNA sequence(s) that bind to one or more adaptor proteins,
and wherein the adaptor protein further recruits one or more
heterologous functional domains. In an embodiment of the invention
the DD-CRISPR enzyme comprises one or two or more mutations in a
residue selected from the group comprising, consisting essentially
of, or consisting of D10, E762, H840, N854, N863, or D986. In a
further embodiment the DD-CRISPR enzyme comprises one or two or
more mutations selected from the group comprising D10A, E762A,
H840A, N854A, N863A or D986A. In another embodiment, the functional
domain comprise, consist essentially of a transcriptional
activation domain, e.g., VP64. In another embodiment, the
functional domain comprise, consist essentially of a
transcriptional repressor domain, e.g., KRAB domain, SID domain or
a SID4X domain. In embodiments of the invention, the one or more
heterologous functional domains have one or more activities
selected from the group comprising, consisting essentially of, or
consisting of methylase activity, demethylase activity,
transcription activation activity, transcription repression
activity, transcription release factor activity, histone
modification activity, RNA cleavage activity and nucleic acid
binding activity. In further embodiments of the invention the cell
is a eukaryotic cell or a mammalian cell or a human cell. In
further embodiments, the adaptor protein is selected from the group
comprising, consisting essentially of, or consisting of MS2, PP7,
Q.beta., F2, GA, fr, JP501, M12, R17, BZ13, JP34, JP500, KU1, M11,
MX1, TW18, VK, SP, FI, ID2, NL95, TW19, AP205, .phi.Cb5, .phi.Cb8r,
.phi.Cb12r, .phi.Cb23r, 7s, PRR1. In another embodiment, the at
least one loop of the sgRNA is tetraloop and/or loop2. An aspect of
the invention encompasses methods of modifying a genomic locus of
interest to change gene expression in a cell by introducing into
the cell any of the compositions described herein.
[0044] An aspect of the invention is that the above elements are
comprised in a single composition or comprised in individual
compositions. These compositions may advantageously be applied to a
host to elicit a functional effect on the genomic level.
[0045] In general, the sgRNA are modified in a manner that provides
specific binding sites (e.g., aptamers) for adapter proteins
comprising one or more functional domains (e.g., via fusion
protein) to bind to. The modified sgRNA are modified such that once
the sgRNA forms a DD-CRISPR complex (i.e. DD-CRISPR enzyme binding
to sgRNA and target) the adapter proteins bind and, the functional
domain on the adapter protein is positioned in a spatial
orientation which is advantageous for the attributed function to be
effective. For example, if the functional domain comprise, consist
essentially of a transcription activator (e.g., VP64 or p65), the
transcription activator is placed in a spatial orientation which
allows it to affect the transcription of the target. Likewise, a
transcription repressor will be advantageously positioned to affect
the transcription of the target and a nuclease (e.g., Fok1) will be
advantageously positioned to cleave or partially cleave the
target.
[0046] The skilled person will understand that modifications to the
sgRNA which allow for binding of the adapter+functional domain but
not proper positioning of the adapter+functional domain (e.g., due
to steric hindrance within the three dimensional structure of the
CRISPR complex) are modifications which are not intended. The one
or more modified sgRNA may be modified at the tetra loop, the stem
loop 1, stem loop 2, or stem loop 3, as described herein,
preferably at either the tetra loop or stem loop 2, and most
preferably at both the tetra loop and stem loop 2.
[0047] As explained herein the functional domains may be, for
example, one or more domains from the group comprising, consisting
essentially of, or consisting of methylase activity, demethylase
activity, transcription activation activity, transcription
repression activity, transcription release factor activity, histone
modification activity, RNA cleavage activity, DNA cleavage
activity, nucleic acid binding activity, and molecular switches
(e.g., light inducible). In some cases it is advantageous that
additionally at least one NLS and/or NES is provided. In some
instances, it is advantageous to position the NLS and/or NES at the
N terminus. When more than one functional domain is included, the
functional domains may be the same or different.
[0048] The sgRNA may be designed to include multiple binding
recognition sites (e.g., aptamers) specific to the same or
different adapter protein. The sgRNA may be designed to bind to the
promoter region -1000-+1 nucleic acids upstream of the
transcription start site (i.e. TSS), preferably -200 nucleic acids.
This positioning improves functional domains which affect gene
activation (e.g., transcription activators) or gene inhibition
(e.g., transcription repressors). The modified sgRNA may be one or
more modified sgRNAs targeted to one or more target loci (e.g., at
least 1 sgRNA, at least 2 sgRNA, at least 5 sgRNA, at least 10
sgRNA, at least 20 sgRNA, at least 30 sg RNA, at least 50 sgRNA)
comprised in a composition.
[0049] Further, the DD-CRISPR enzyme with diminished nuclease
activity is most effective when the nuclease activity is
inactivated (e.g., nuclease inactivation of at least 70%, at least
80%, at least 90%, at least 95%, at least 97%, or 100% as compared
with the wild type enzyme; or to put in another way, a DD-Cas9
enzyme or DD-CRISPR enzyme having advantageously about 0% of the
nuclease activity of the non-mutated or wild type Cas9 enzyme or
CRISPR enzyme, or no more than about 3% or about 5% or about 10% of
the nuclease activity of the non-mutated or wild type Cas9 enzyme
or CRISPR enzyme). This is possible by introducing mutations into
the RuvC and HNH nuclease domains of the SpCas9 and orthologs
thereof. For example utilizing mutations in a residue selected from
the group comprising, consisting essentially of, or consisting of
D10, E762, H840, N854, N863, or D986 and more preferably
introducing one or more of the mutations selected from the group
comprising, consisting essentially of, or consisting of D10A,
E762A, H840A, N854A, N863A or D986A. A preferable pair of mutations
is D10A with H840A, more preferable is D10A with N863A of SpCas9
and orthologs thereof. The inactivated CRISPR enzyme may have
associated (e.g., via fusion protein) one or more functional
domains, e.g., at least one destabilizing domain; or, for instance
like those as described herein for the modified sgRNA adaptor
proteins, including for example, one or more domains from the group
comprising, consisting essentially of, or consisting of methylase
activity, demethylase activity, transcription activation activity,
transcription repression activity, transcription release factor
activity, histone modification activity, RNA cleavage activity, DNA
cleavage activity, nucleic acid binding activity, and molecular
switches (e.g., light inducible). Preferred domains are Fok1, VP64,
P65, HSF1, MyoD1. In the event that Fok1 is provided, it is
advantageous that multiple Fok1 functional domains are provided to
allow for a functional dimer and that sgRNAs are designed to
provide proper spacing for functional use (Fok1) as specifically
described in Tsai et al. Nature Biotechnology, Vol. 32, Number 6,
June 2014). The adaptor protein may utilize known linkers to attach
such functional domains. In some cases it is advantageous that
additionally at least one NLS or NES is provided. In some
instances, it is advantageous to position the NLS or NES at the N
terminus. When more than one functional domain is included, the
functional domains may be the same or different. In general, the
positioning of the one or more functional domain on the inactivated
DD-CRISPR enzyme is one which allows for correct spatial
orientation for the functional domain to affect the target with the
attributed functional effect. For example, if the functional domain
is a transcription activator (e.g., VP64 or p65), the transcription
activator is placed in a spatial orientation which allows it to
affect the transcription of the target. Likewise, a transcription
repressor will be advantageously positioned to affect the
transcription of the target, and a nuclease (e.g., Fok1) will be
advantageously positioned to cleave or partially cleave the target.
This may include positions other than the N-/C-terminus of the
DD-CRISPR enzyme. Positioning the functional domain in the Rec1
domain, the Rec2 domain, the HNH domain, or the PI domain of the
SpCas9 protein or any ortholog corresponding to these domains is
advantageous; and again, it is mentioned that the functional domain
can be a DD. Positioning of the functional domains to the Rec1
domain or the Rec2 domain, of the SpCas9 protein or any ortholog
corresponding to these domains, in some instances may be preferred.
Positioning of the functional domains to the Rec1 domain at
position 553, Rec1 domain at 575, the Rec2 domain at any position
of 175-306 or replacement thereof, the HNH domain at any position
of 715-901 or replacement thereof, or the PI domain at position
1153 of the SpCas9 protein or any ortholog corresponding to these
domains, in some instances may be preferred. Fok1 functional domain
may be attached at the N terminus. When more than one functional
domain is included, the functional domains may be the same or
different.
[0050] An adaptor protein may be any number of proteins that binds
to an aptamer or recognition site introduced into the modified
sgRNA and which allows proper positioning of one or more functional
domains, once the sgRNA has been incorporated into the DD-CRISPR
complex, to affect the target with the attributed function. As
explained in detail in this application such may be coat proteins,
preferably bacteriophage coat proteins. The functional domains
associated with such adaptor proteins (e.g., in the form of fusion
protein) may include, for example, one or more domains from the
group comprising, consisting essentially of, or consisting of
methylase activity, demethylase activity, transcription activation
activity, transcription repression activity, transcription release
factor activity, histone modification activity, RNA cleavage
activity, DNA cleavage activity, nucleic acid binding activity, and
molecular switches (e.g., light inducible). Preferred domains are
Fok1, VP64, P65, HSF1, MyoD1. In the event that the functional
domain is a transcription activator or transcription repressor it
is advantageous that additionally at least an NLS or NES is
provided and preferably at the N terminus. When more than one
functional domain is included, the functional domains may be the
same or different. The adaptor protein may utilize known linkers to
attach such functional domains. Such linkers may be used to
associate the DD with the CRISPR enzyme or have the CRISPR enzyme
comprise the DD.
[0051] Thus, sgRNA, e.g., modified sgRNA, the inactivated DD-CRISPR
enzyme (with or without functional domains), and the binding
protein with one or more functional domains, may each individually
be comprised in a composition and administered to a host
individually or collectively. Alternatively, these components may
be provided in a single composition for administration to a host.
Administration to a host may be performed via viral vectors known
to the skilled person or described herein for delivery to a host
(e.g., lentiviral vector, adenoviral vector, AAV vector). As
explained herein, use of different selection markers (e.g., for
lentiviral sgRNA selection) and concentration of sgRNA (e.g.,
dependent on whether multiple sgRNAs are used) may be advantageous
for eliciting an improved effect. On the basis of this concept,
several variations are appropriate to elicit a genomic locus event,
including DNA cleavage, gene activation, or gene deactivation.
Using the provided compositions, the person skilled in the art can
advantageously and specifically target single or multiple loci with
the same or different functional domains to elicit one or more
genomic locus events. The compositions may be applied in a wide
variety of methods for screening in libraries in cells and
functional modeling in vivo (e.g., gene activation of lincRNA and
identification of function; gain-of-function modeling;
loss-of-function modeling; the use the compositions of the
invention to establish cell lines and transgenic animals for
optimization and screening purposes).
[0052] The current invention comprehends the use of the
compositions of the current invention to establish and utilize
conditional or inducible DD-CRISPR transgenic cell/animals; see,
e.g., Platt et al., Cell (2014), 159(2): 440-455, or PCT patent
publications cited herein, such as WO 2014/093622
(PCT/US2013/074667). For example, cells or animals such as
non-human animals, e.g., vertebrates or mammals, such as rodents,
e.g., mice, rats, or other laboratory or field animals, e.g., cats,
dogs, sheep, etc., may be `knock-in` whereby the animal
conditionally or inducibly expresses DD-Cas9 akin to Platt et al.
The target cell or animal thus comprises DD-CRISPR enzyme (e.g.,
DD-Cas9) conditionally or inducibly (e.g., in the form of Cre
dependent constructs) and/or the adapter protein or DD
conditionally or inducibly and, on expression of a vector
introduced into the target cell, the vector expresses that which
induces or gives rise to the condition of DD-CRISPR enzyme (e.g.,
DD-Cas9) expression and/or adaptor or DD expression in the target
cell. By applying the teaching and compositions of the current
invention with the known method of creating a CRISPR complex,
inducible genomic events are also an aspect of the current
invention. One mere example of this is the creation of a CRISPR
knock-in/conditional transgenic animal (e.g., mouse comprising
e.g., a Lox-Stop-polyA-Lox(LSL) cassette) and subsequent delivery
of one or more compositions providing one or more modified sgRNA
(e.g., -200 nucleotides to TSS of a target gene of interest for
gene activation purposes, e.g., modified sgRNA with one or more
aptamers recognized by coat proteins, e.g., MS2), one or more
adapter proteins as described herein (MS2 binding protein linked to
one or more VP64) and means for inducing the conditional animal
(e.g., Cre recombinase for rendering DD-Cas9 expression inducible).
Alternatively, the adaptor protein or DD may be provided as a
conditional or inducible element with a conditional or inducible
CRISPR enzyme to provide an effective model for screening purposes,
which advantageously only requires minimal design and
administration of specific sgRNAs for a broad number of
applications.
[0053] In some embodiments, phenotypic alteration is preferably the
result of genome modification when a genetic disease is targeted,
especially in methods of therapy and preferably where a repair
template is provided to correct or alter the phenotype.
[0054] In some embodiments diseases that may be targeted include
those concerned with disease-causing splice defects.
[0055] In some embodiments, cellular targets include Hemopoietic
Stem/Progenitor Cells (CD34+); Human T cells; and Eye (retinal
cells)--for example photoreceptor precursor cells.
[0056] In some embodiments Gene targets include: Human Beta
Globin--HBB (for treating Sickle Cell Anemia, including by
stimulating gene-conversion (using closely related HBD gene as an
endogenous template)); CD3 (T-Cells); and CEP920--retina (eye).
[0057] In some embodiments disease targets also include: cancer;
Sickle Cell Anemia (based on a point mutation); HBV, HIV;
Beta-Thalassemia; and ophthalmic or ocular disease--for example
Leber Congenital Amaurosis (LCA)-causing Splice Defect.
[0058] In some embodiments delivery methods include: Cationic Lipid
Mediated "direct" delivery of Enzyme-Guide complex
(RiboNucleoProtein) and electroporation of plasmid DNA.
[0059] Methods, products and uses described herein may be used for
non-therapeutic purposes. Furthermore, any of the methods described
herein may be applied in vitro and ex vivo.
[0060] In an aspect, provided is a non-naturally occurring or
engineered composition comprising:
[0061] I. two or more CRISPR-Cas system polynucleotide sequences
comprising
[0062] (a) a first guide sequence capable of hybridizing to a first
target sequence in a polynucleotide locus,
[0063] (b) a second guide sequence capable of hybridizing to a
second target sequence in a polynucleotide locus,
[0064] (c) a tracr mate sequence, and
[0065] (d) a tracrRNA sequence, and
[0066] II. a Type II Cas9 enzyme or a second polynucleotide
sequence encoding it,
[0067] wherein the Type II Cas9 enzyme is a modified enzyme
comprising one or more DD as described herein,
[0068] wherein when transcribed, the first and the second tracr
mate sequences hybridize to the first and second tracrRNA sequences
respectively and the first and the second guide sequences direct
sequence-specific binding of a first and a second CRISPR complex to
the first and second target sequences respectively,
[0069] wherein the first CRISPR complex comprises the Cas9 enzyme
complexed with (1) the first guide sequence that is hybridizable to
the first target sequence, and (2) the first tracr mate sequence
that is hybridized to the first tracrRNA sequence,
[0070] wherein the second CRISPR complex comprises the Cas9 enzyme
complexed with (1) the second guide sequence that is hybridizable
to the second target sequence, and (2) the second tracr mate
sequence that is hybridized to the second tracrRNA sequence,
and
[0071] wherein the first guide sequence directs cleavage of one
strand of the DNA duplex near the first target sequence and the
second guide sequence directs cleavage of the other strand near the
second target sequence inducing a double strand break, thereby
modifying the organism or the non-human or non-animal organism.
[0072] In another embodiment, the Cas9 is delivered into the cell
as a protein. In another and particularly preferred embodiment, the
Cas9 is delivered into the cell as a protein or as a nucleotide
sequence encoding it. Delivery to the cell as a protein may include
delivery of a Ribonucleoprotein (RNP) complex, where the protein is
complexed with the guide.
[0073] In some embodiments, the ortholog is Staphylococcus aureus
so that the Cas9 is that from or derived from Staphylococcus aureus
(referred to as SaCas9). In some embodiments, the Staphylococcus
aureus is Staphylococcus aureus subspecies aureus. Guidance is
provided below in respect of guide length (the spacer or guide
sequence). In some embodiments, for Sp, optimal guide length can
vary as low as a 17-nucleotides or what is known in the art as a
tru-guide or tru-sgRNAs (see, e.g., Fu et al., "Improving
CRISPR-Cas nuclease specificity using truncated guide RNAs," Nature
Biotechnology 32, 279-284 (2014) doi:10.1038/nbt.2808 Received 17
Nov. 2013 Accepted 6 Jan. 2014 Published online 26 Jan. 2014
Corrected online 29 Jan. 2014) In some embodiments, for Sa, the
optimal guide length may be 19, 20 or 21 or 22 or 23 or 24
nucleotides in length (Ran et al. (2015), mentioned below).
[0074] In an aspect, host cells and cell lines modified by or
comprising the compositions, systems or modified enzymes of present
invention are provided, including stem cells, and progeny
thereof.
[0075] In an aspect, methods of cellular therapy are provided,
where, for example, a single cell or a population of cells is
sampled or cultured, wherein that cell or cells is or has been
modified ex vivo as described herein, and is then re-introduced
(sampled cells) or introduced (cultured cells) into the organism.
Stem cells, whether embryonic or induce pluripotent or totipotent
stem cells, are also particularly preferred in this regard. But, of
course, in vivo embodiments are also envisaged.
[0076] Inventive methods can further comprise delivery of
templates, such as repair templates, which may be dsODN or ssODN,
see below. Delivery of templates may be via the cotemporaneous or
separate from delivery of any or all the CRISPR enzyme, guide,
tracr mate or tracrRNA and via the same delivery mechanism or
different. In some embodiments, it is preferred that the template
is delivered together with the guide, tracr mate and/or tracrRNA
and, preferably, also the CRISPR enzyme. An example may be an AAV
vector where the CRISPR enzyme is SaCas9 (with the N580
mutation).
[0077] Inventive methods can further comprise: (a) delivering to
the cell a double-stranded oligodeoxynucleotide (dsODN) comprising
overhangs complimentary to the overhangs created by said double
strand break, wherein said dsODN is integrated into the locus of
interest; or--(b) delivering to the cell a single-stranded
oligodeoxynucleotide (ssODN), wherein said ssODN acts as a template
for homology directed repair of said double strand break. Inventive
methods can be for the prevention or treatment of disease in an
individual, optionally wherein said disease is caused by a defect
in said locus of interest. Inventive methods can be conducted in
vivo in the individual or ex vivo on a cell taken from the
individual, optionally wherein said cell is returned to the
individual.
[0078] The tracr sequence may be referred to as the tracrRNA. In
some embodiments, it may be at least 30, at least 40 or at least 50
nucleotides in length.
[0079] The invention also comprehends products obtained from using
CRISPR enzyme or Cas enzyme or Cas9 enzyme or CRISPR-CRISPR enzyme
or CRISPR-Cas system or CRISPR-Cas9 system of the invention.
[0080] Accordingly, it is an object of the invention not to
encompass within the invention any previously known product,
process of making the product, or method of using the product such
that Applicants reserve the right and hereby disclose a disclaimer
of any previously known product, process, or method. It is further
noted that the invention does not intend to encompass within the
scope of the invention any product, process, or making of the
product or method of using the product, which does not meet the
written description and enablement requirements of the USPTO (35
U.S.C. .sctn.112, first paragraph) or the EPO (Article 83 of the
EPC), such that Applicants reserve the right and hereby disclose a
disclaimer of any previously described product, process of making
the product, or method of using the product. It may be advantageous
in the practice of the invention to be in compliance with Art.
53(c) EPC and Rule 28(b) and (c) EPC. Nothing herein is to be
construed as a promise.
[0081] It is noted that in this disclosure and particularly in the
claims and/or paragraphs, terms such as "comprises", "comprised",
"comprising" and the like can have the meaning attributed to it in
U.S. patent law; e.g., they can mean "includes", "included",
"including", and the like; and that terms such as "consisting
essentially of" and "consists essentially of" have the meaning
ascribed to them in U.S. patent law, e.g., they allow for elements
not explicitly recited, but exclude elements that are found in the
prior art or that affect a basic or novel characteristic of the
invention.
[0082] These and other embodiments are disclosed or are obvious
from and encompassed by, the following Detailed Description.
BRIEF DESCRIPTION OF THE DRAWINGS
[0083] The novel features of the invention are set forth with
particularity in the appended claims. A better understanding of the
features and advantages of the present invention will be obtained
by reference to the following detailed description that sets forth
illustrative embodiments, in which the principles of the invention
are utilized, and the accompanying drawings of which:
[0084] FIG. 1 shows indels detected by deep sequencing in HEK293FT
cells transfected with 400 ng ER50-Cas9, Cas9-ER50 or
ER50-Cas9-ER50 and 100 ng PCR amplified U6-sgRNA targeting EMX1.
ER50 was stabilized with either 4-hydroxytamoxifen (4HT) or CMP8.
Two different concentrations were tested for each ligand.
[0085] FIG. 2 shows indels detected by deep sequencing in HEK293FT
cells transfected with 400 ng DHFR-Cas9, Cas9-DHFR or
DHFR-Cas9-DHFR and 100 ng PCR amplified U6-sgRNA targeting EMX1.
ER50 was stabilized with trimethoprim (TMP). Two different
concentrations were tested.
[0086] FIG. 3 shows when ER50 or DHFR was fused to the C-term of
FKBP(C)Cas9-2.times.NLS, indels were detected by deep sequencing in
HEK293FT cells transfected with 200 ng FRB(N)Cas9-NLS,
FRB(N)Cas9-noNLS or FRB(N)Cas9-NES and with 200 ng
FKBP(C)Cas9-2.times.NLS-DD and 100 ng PCR amplified U6-sgRNA
targeting EMX1. ER50 fusions were treated with either 10 uM 4HT or
1.5 uM CMP8. DHFR fusions were treated with 10 uM TMP.
[0087] FIG. 4 shows a comparison of two SpCas9-DD fusions:
SpCas9-DHFR and SpCas9-ER50. Both were direct fusions of the DD to
the C' terminal of SpCas9. pSAM88 is a control. GFP is a marker. A
single DD was used in each fusion to the SpCas9. The DHFR DD showed
an approximately 2 fold increase in activity in the presence of its
stabilizing ligand, TMP, compared to in the absence of TMP. The
ER50 DD showed an approximately 12-13 fold increase in activity in
the presence of its stabilizing ligand, 4HT, compared to in the
absence of said ligand. Thus, although DHFR is useful with SpCas9,
ER50 seems to be particularly useful with respect to controlling
activity of SpCas9 as there was a large increase in activity in the
presence of the stabilizing ligand for the ER50 DD.
[0088] FIG. 5 shows ER50-dSaCas9-VP64 activation of ASCL1.
Destabilized dSaCas9-VP64 in combination with another activator
(activator described elsewhere which does not contain a
destabilized domain). MS2 recognition loops were added to the
SaCas9 sgRNA scaffold in a similar manner to the SpCas9 sgRNA
scaffold.
[0089] FIG. 6 shows a dose dependent decrease in EGFP positive
cells treated with SpCas9-DD fusion protein and increasing dosages
of the DD-domain ligand trimethoprim (TMP). The SpCas9-DD protein
(DHFR-SpCas9-DHFR; "DSPD") was provided to an EGFP-producing cell
line with a guide RNA targeted to EGFP gene. EGFP fluorescence was
reduced to about 60-80% of the untreated level by the SpCas9-DD
protein alone, depending on the dose. Addition of the DHFR ligand
trimethoprim (TMP) increased the effect of the SpCas9-DD fusion
protein in a dose-dependent manner.
[0090] FIG. 7 shows a dose dependent decrease in EGFP positive
cells treated with SpCas9-DD fusion protein and increasing dosages
of the DD-domain ligand 4HT. The SpCas9-DD protein
(ER50-SpCas9-ER50; "ESPE") was provided to an EGFP-producing cell
line with a guide RNA targeted to EGFP gene. EGFP fluorescence was
reduced to about 70-80% of the untreated level by the SpCas9-DD
protein alone, depending on the dose. Addition of the ER50 ligand
4HT increased the effect of the SpCas9-DD fusion protein in a
dose-dependent manner.
[0091] The figures herein are for illustrative purposes only and
are not necessarily drawn to scale.
DETAILED DESCRIPTION OF THE INVENTION
[0092] As shown herein, for example exemplified in Example 1, the
DHFR DD showed an approximately 2 fold increase in activity in the
presence of its stabilizing ligand, TMP, compared to in the absence
of TMP. The ER50 DD showed an approximately 12-13 fold increase in
activity in the presence of its stabilizing ligand, 4HT, compared
to in the absence of said ligand. Thus, although DHFR is useful
with SpCas9, ER50 seems to be particularly useful with respect to
controlling activity of SpCas9 as there was a large increase in
activity in the presence of the stabilizing ligand for the ER50 DD,
see FIGS. 1-3.
[0093] Example 1 was repeated with a Cas9 from Staphylococcus
aureus. The results with activation of transcription of the ASCL1
gene into ASCL1 mRNA are shown in FIG. 4. The DD used are DHFR and
ER50, as before. The stabilizing ligands are TMP and 4HT,
respectively, as before. Significant reductions in transcription
are seen in the absence of each stabilizing ligand compared to in
the presence of each ligand (for the corresponding DD). This
provides further and useful validation of the results in Example 1
in an ortholog of SpCas9, SaCas9.
[0094] In general, the CRISPR-Cas, CRISPR-Cas9 or CRISPR system is
as used in the foregoing documents, such as WO 2014/093622
(PCT/US2013/074667) and refers collectively to transcripts and
other elements involved in the expression of or directing the
activity of CRISPR-associated ("Cas") genes, including sequences
encoding a Cas gene, in particular a Cas9 gene in the case of
CRISPR-Cas9, a tracr (trans-activating CRISPR) sequence (e.g.,
tracrRNA or an active partial tracrRNA), a tracr-mate sequence
(encompassing a "direct repeat" and a tracrRNA-processed partial
direct repeat in the context of an endogenous CRISPR system), a
guide sequence (also referred to as a "spacer" in the context of an
endogenous CRISPR system), or "RNA(s)" as that term is herein used
(e.g., RNA(s) to guide Cas9, e.g., CRISPR RNA and transactivating
(tracr) RNA or a single guide RNA (sgRNA) (chimeric RNA)) or other
sequences and transcripts from a CRISPR locus. In general, a CRISPR
system is characterized by elements that promote the formation of a
CRISPR complex at the site of a target sequence (also referred to
as a protospacer in the context of an endogenous CRISPR system). In
the context of formation of a CRISPR complex, "target sequence"
refers to a sequence to which a guide sequence is designed to have
complementarity, where hybridization between a target sequence and
a guide sequence promotes the formation of a CRISPR complex. A
target sequence may comprise any polynucleotide, such as DNA or RNA
polynucleotides. In some embodiments, a target sequence is located
in the nucleus or cytoplasm of a cell, and may include nucleic
acids in or from mitochondrial, organelles, vesicles, liposomes or
particles present within the cell. In some embodiments, especially
for non-nuclear uses, NLSs are not preferred. In some embodiments,
direct repeats may be identified in silico by searching for
repetitive motifs that fulfill any or all of the following
criteria: 1. found in a 2 Kb window of genomic sequence flanking
the type II CRISPR locus; 2. span from 20 to 50 bp; and 3.
interspaced by 20 to 50 bp. In some embodiments, 2 of these
criteria may be used, for instance 1 and 2, 2 and 3, or 1 and 3. In
some embodiments, all 3 criteria may be used.
[0095] In embodiments of the invention the terms guide sequence and
guide RNA are used interchangeably as in foregoing cited documents
such as WO 2014/093622 (PCT/US2013/074667). In general, a guide
sequence is any polynucleotide sequence having sufficient
complementarity with a target polynucleotide sequence to hybridize
with the target sequence and direct sequence-specific binding of a
CRISPR complex to the target sequence. In some embodiments, the
degree of complementarity between a guide sequence and its
corresponding target sequence, when optimally aligned using a
suitable alignment algorithm, is about or more than about 50%, 60%,
75%, 80%, 85%, 90%, 95%, 97.5%, 99%, or more. Optimal alignment may
be determined with the use of any suitable algorithm for aligning
sequences, non-limiting example of which include the Smith-Waterman
algorithm, the Needleman-Wunsch algorithm, algorithms based on the
Burrows-Wheeler Transform (e.g., the Burrows Wheeler Aligner),
ClustalW, Clustal X, BLAT, Novoalign (Novocraft Technologies;
available at www.novocraft.com), ELAND (Illumina, San Diego,
Calif.), SOAP (available at soap.genomics.org.cn), and Maq
(available at maq.sourceforge.net). In some embodiments, a guide
sequence is about or more than about 5, 10, 11, 12, 13, 14, 15, 16,
17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 35, 40, 45,
50, 75, or more nucleotides in length. In some embodiments, a guide
sequence is less than about 75, 50, 45, 40, 35, 30, 25, 20, 15, 12,
or fewer nucleotides in length. Preferably the guide sequence is
10-30 nucleotides long. The ability of a guide sequence to direct
sequence-specific binding of a CRISPR complex to a target sequence
may be assessed by any suitable assay. For example, the components
of a CRISPR system sufficient to form a CRISPR complex, including
the guide sequence to be tested, may be provided to a host cell
having the corresponding target sequence, such as by transfection
with vectors encoding the components of the CRISPR sequence,
followed by an assessment of preferential cleavage within the
target sequence, such as by Surveyor assay as described herein.
Similarly, cleavage of a target polynucleotide sequence may be
evaluated in a test tube by providing the target sequence,
components of a CRISPR complex, including the guide sequence to be
tested and a control guide sequence different from the test guide
sequence, and comparing binding or rate of cleavage at the target
sequence between the test and control guide sequence reactions.
Other assays are possible, and will occur to those skilled in the
art.
[0096] In a classic CRISPR-Cas system, the degree of
complementarity between a guide sequence and its corresponding
target sequence can be about or more than about 50%, 60%, 75%, 80%,
85%, 90%, 95%, 97.5%, 99%, or 100%; a guide or RNA or sgRNA can be
about or more than about 5, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19,
20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 35, 40, 45, 50, 75, or
more nucleotides in length; or guide or RNA or sgRNA can be less
than about 75, 50, 45, 40, 35, 30, 25, 20, 15, 12, or fewer
nucleotides in length; and advantageously tracr RNA is 30 or 50
nucleotides in length. However, an aspect of the invention is to
reduce off-target interactions, e.g., reduce the guide interacting
with a target sequence having low complementarity. Indeed, in the
examples, it is shown that the invention involves mutations that
result in the CRISPR-Cas system being able to distinguish between
target and off-target sequences that have greater than 80% to about
95% complementarity, e.g., 83%-84% or 88-89% or 94-95%
complementarity (for instance, distinguishing between a target
having 18 nucleotides from an off-target of 18 nucleotides having
1, 2 or 3 mismatches). Accordingly, in the context of the present
invention the degree of complementarity between a guide sequence
and its corresponding target sequence is greater than 94.5% or 95%
or 95.5% or 96% or 96.5% or 97% or 97.5% or 98% or 98.5% or 99% or
99.5% or 99.9%, or 100%. Off target is less than 100% or 99.9% or
99.5% or 99% or 99% or 98.5% or 98% or 97.5% or 97% or 96.5% or 96%
or 95.5% or 95% or 94.5% or 94% or 93% or 92% or 91% or 90% or 89%
or 88% or 87% or 86% or 85% or 84% or 83% or 82% or 81% or 80%
complementarity between the sequence and the guide, with it
advantageous that off target is 100% or 99.9% or 99.5% or 99% or
99% or 98.5% or 98% or 97.5% or 97% or 96.5% or 96% or 95.5% or 95%
or 94.5% complementarity between the sequence and the guide.
[0097] In particularly preferred embodiments according to the
invention, the protected guide RNA (capable of guiding Cas to a
target locus) may comprise (1) a guide sequence (including its
protector sequence) capable of hybridizing to a genomic target
locus in the eukaryotic cell; (2) a tracr sequence; and (3) a tracr
mate sequence. All (1) to (3) may reside in a single RNA, i.e. an
sgRNA (arranged in a 5' to 3' orientation), or the tracr RNA may be
a different RNA than the RNA containing the guide and tracr
sequence. The tracr hybridizes to the tracr mate sequence and
directs the CRISPR/Cas complex to the target sequence.
[0098] The methods according to the invention as described herein
comprehend inducing one or more mutations in a eukaryotic cell (in
vitro, i.e. in an isolated eukaryotic cell) as herein discussed
comprising delivering to cell a vector as herein discussed. The
mutation(s) can include the introduction, deletion, or substitution
of one or more nucleotides at each target sequence of cell(s) via
the guide(s) RNA(s) or sgRNA(s). The mutations can include the
introduction, deletion, or substitution of 1-75 nucleotides at each
target sequence of said cell(s) via the guide(s) RNA(s) or
sgRNA(s). The mutations can include the introduction, deletion, or
substitution of 1, 5, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20,
21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 35, 40, 45, 50, or 75
nucleotides at each target sequence of said cell(s) via the
guide(s) RNA(s) or sgRNA(s). The mutations can include the
introduction, deletion, or substitution of 5, 10, 11, 12, 13, 14,
15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 35,
40, 45, 50, or 75 nucleotides at each target sequence of said
cell(s) via the guide(s) RNA(s) or sgRNA(s). The mutations include
the introduction, deletion, or substitution of 10, 11, 12, 13, 14,
15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 35,
40, 45, 50, or 75 nucleotides at each target sequence of said
cell(s) via the guide(s) RNA(s) or sgRNA(s). The mutations can
include the introduction, deletion, or substitution of 20, 21, 22,
23, 24, 25, 26, 27, 28, 29, 30, 35, 40, 45, 50, or 75 nucleotides
at each target sequence of said cell(s) via the guide(s) RNA(s) or
sgRNA(s). The mutations can include the introduction, deletion, or
substitution of 40, 45, 50, 75, 100, 200, 300, 400 or 500
nucleotides at each target sequence of said cell(s) via the
guide(s) RNA(s) or sgRNA(s).
[0099] For minimization of toxicity and off-target effect, it will
be important to control the concentration of Cas mRNA and guide RNA
delivered. Optimal concentrations of Cas mRNA and guide RNA can be
determined by testing different concentrations in a cellular or
non-human eukaryote animal model and using deep sequencing the
analyze the extent of modification at potential off-target genomic
loci. Alternatively, to minimize the level of toxicity and
off-target effect, Cas nickase mRNA (for example S. pyogenes Cas9
with the D10A mutation) can be delivered with a pair of guide RNAs
targeting a site of interest. Guide sequences and strategies to
minimize toxicity and off-target effects can be as in WO
2014/093622 (PCT/US2013/074667); or, via mutation as herein.
[0100] Typically, in the context of an endogenous CRISPR system,
formation of a CRISPR complex (comprising a guide sequence
hybridized to a target sequence and complexed with one or more Cas
proteins) results in cleavage of one or both strands in or near
(e.g. within 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 50, or more base
pairs from) the target sequence. Without wishing to be bound by
theory, the tracr sequence, which may comprise or consist of all or
a portion of a wild-type tracr sequence (e.g. about or more than
about 20, 26, 32, 45, 48, 54, 63, 67, 85, or more nucleotides of a
wild-type tracr sequence), may also form part of a CRISPR complex,
such as by hybridization along at least a portion of the tracr
sequence to all or a portion of a tracr mate sequence that is
operably linked to the guide sequence.
[0101] The nucleic acid molecule encoding a Cas9 is advantageously
codon optimized Cas9. An example of a codon optimized sequence, is
in this instance a sequence optimized for expression in a
eukaryote, e.g., humans (i.e. being optimized for expression in
humans), or for another eukaryote, animal or mammal as herein
discussed; see, e.g., SaCas9 human codon optimized sequence in WO
2014/093622 (PCT/US2013/074667). Whilst this is preferred, it will
be appreciated that other examples are possible and codon
optimization for a host species other than human, or for codon
optimization for specific organs is known. In some embodiments, an
enzyme coding sequence encoding a Cas9 is codon optimized for
expression in particular cells, such as eukaryotic cells. The
eukaryotic cells may be those of or derived from a particular
organism, such as a mammal, including but not limited to human, or
non-human eukaryote or animal or mammal as herein discussed, e.g.,
mouse, rat, rabbit, dog, livestock, or non-human mammal or primate.
In some embodiments, processes for modifying the germ line genetic
identity of human beings and/or processes for modifying the genetic
identity of animals which are likely to cause them suffering
without any substantial medical benefit to man or animal, and also
animals resulting from such processes, may be excluded. In general,
codon optimization refers to a process of modifying a nucleic acid
sequence for enhanced expression in the host cells of interest by
replacing at least one codon (e.g. about or more than about 1, 2,
3, 4, 5, 10, 15, 20, 25, 50, or more codons) of the native sequence
with codons that are more frequently or most frequently used in the
genes of that host cell while maintaining the native amino acid
sequence. Various species exhibit particular bias for certain
codons of a particular amino acid. Codon bias (differences in codon
usage between organisms) often correlates with the efficiency of
translation of messenger RNA (mRNA), which is in turn believed to
be dependent on, among other things, the properties of the codons
being translated and the availability of particular transfer RNA
(tRNA) molecules. The predominance of selected tRNAs in a cell is
generally a reflection of the codons used most frequently in
peptide synthesis. Accordingly, genes can be tailored for optimal
gene expression in a given organism based on codon optimization.
Codon usage tables are readily available, for example, at the
"Codon Usage Database" available at www.kazusa.orjp/codon/ and
these tables can be adapted in a number of ways. See Nakamura, Y.,
et al. "Codon usage tabulated from the international DNA sequence
databases: status for the year 2000" Nucl. Acids Res. 28:292
(2000). Computer algorithms for codon optimizing a particular
sequence for expression in a particular host cell are also
available, such as Gene Forge (Aptagen; Jacobus, Pa.), are also
available. In some embodiments, one or more codons (e.g. 1, 2, 3,
4, 5, 10, 15, 20, 25, 50, or more, or all codons) in a sequence
encoding a Cas9 correspond to the most frequently used codon for a
particular amino acid.
[0102] In certain embodiments, the methods as described herein may
comprise providing a Cas9 transgenic cell in which one or more
nucleic acids encoding one or more guide RNAs are provided or
introduced operably connected in the cell with a regulatory element
comprising a promoter of one or more gene of interest. As used
herein, the term "Cas transgenic cell" refers to a cell, such as a
eukaryotic cell, in which a Cas9 gene has been genomically
integrated. The nature, type, or origin of the cell are not
particularly limiting according to the present invention. Also the
way how the Cas9 transgene is introduced in the cell is may vary
and can be any method as is known in the art. In certain
embodiments, the Cas9 transgenic cell is obtained by introducing
the Cas9 transgene in an isolated cell. In certain other
embodiments, the Cas transgenic cell is obtained by isolating cells
from a Cas9 transgenic organism. By means of example, and without
limitation, the Cas9 transgenic cell as referred to herein may be
derived from a Cas transgenic eukaryote, such as a Cas9 knock-in
eukaryote. Reference is made to WO 2014/093622 (PCT/US13/74667),
incorporated herein by reference. Methods of US Patent Publication
Nos. 20120017290 and 20110265198 assigned to Sangamo BioSciences,
Inc. directed to targeting the Rosa locus may be modified to
utilize the CRISPR Cas system of the present invention. Methods of
US Patent Publication No. 20130236946 assigned to Cellectis
directed to targeting the Rosa locus may also be modified to
utilize the CRISPR Cas system of the present invention. By means of
further example reference is made to Platt et. al. (Cell;
159(2):440-455 (2014)), describing a Cas9 knock-in mouse, which is
incorporated herein by reference. The Cas transgene can further
comprise a Lox-Stop-polyA-Lox(LSL) cassette thereby rendering Cas
expression inducible by Cre recombinase. Alternatively, the Cas9
transgenic cell may be obtained by introducing the Cas9 transgene
in an isolated cell. Delivery systems for transgenes are well known
in the art. By means of example, the Cas9 transgene may be
delivered in for instance eukaryotic cell by means of vector (e.g.,
AAV, adenovirus, lentivirus) and/or particle and/or nanoparticle
delivery, as also described herein elsewhere.
[0103] It will be understood by the skilled person that the cell,
such as the Cas9 transgenic cell, as referred to herein may
comprise further genomic alterations besides having an integrated
Cas9 gene or the mutations arising from the sequence specific
action of Cas9 when complexed with RNA capable of guiding Cas9 to a
target locus, such as for instance one or more oncogenic mutations,
as for instance and without limitation described in Platt et al.
(2014), Chen et al., (2014) or Kumar et al. (2009).
[0104] In some embodiments, the Cas9 sequence is fused to one or
more nuclear localization sequences (NLSs), such as about or more
than about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more NLSs. In some
embodiments, the Cas9 comprises about or more than about 1, 2, 3,
4, 5, 6, 7, 8, 9, 10, or more NLSs at or near the amino-terminus,
about or more than about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more
NLSs at or near the carboxy-terminus, or a combination of these
(e.g. zero or at least one or more NLS at the amino-terminus and
zero or at one or more NLS at the carboxy terminus). When more than
one NLS is present, each may be selected independently of the
others, such that a single NLS may be present in more than one copy
and/or in combination with one or more other NLSs present in one or
more copies. In a preferred embodiment of the invention, the Cas9
comprises at most 6 NLSs. In some embodiments, an NLS is considered
near the N- or C-terminus when the nearest amino acid of the NLS is
within about 1, 2, 3, 4, 5, 10, 15, 20, 25, 30, 40, 50, or more
amino acids along the polypeptide chain from the N- or C-terminus.
Non-limiting examples of NLSs include an NLS sequence derived from:
the NLS of the SV40 virus large T-antigen, having the amino acid
sequence PKKKRKV (SEQ ID NO: 1); the NLS from nucleoplasmin (e.g.
the nucleoplasmin bipartite NLS with the sequence KRPAATKKAGQAKKKK)
(SEQ ID NO: 2); the c-myc NLS having the amino acid sequence
PAAKRVKLD (SEQ ID NO: 3) or RQRRNELKRSP (SEQ ID NO: 4); the hRNPA1
M9 NLS having the sequence
NQSSNFGPMKGGNFGGRSSGPYGGGGQYFAKPRNQGGY(SEQ ID NO: 5); the sequence
RMRIZFKNKGKDTAELRRRRVEVSVELRKAKKDEQILKRRNV (SEQ ID NO: 6) of the
IBB domain from importin-alpha; the sequences VSRKRPRP (SEQ ID NO:
7) and PPKKARED (SEQ ID NO: 8) of the myoma T protein; the sequence
PQPKKKPL (SEQ ID NO: 9) of human p53; the sequence SALIKKKKKMAP
(SEQ ID NO: 10) of mouse c-abl IV; the sequences DRLRR (SEQ ID NO:
11) and PKQKKRK (SEQ ID NO: 12) of the influenza virus NS1; the
sequence RKLKKKIKKL (SEQ ID NO: 13) of the Hepatitis virus delta
antigen; the sequence REKKKFLKRR (SEQ ID NO: 14) of the mouse Mx1
protein; the sequence KRKGDEVDGVDEVAKKKSKK (SEQ ID NO: 15) of the
human poly(ADP-ribose) polymerase; and the sequence
RKCLQAGMNLEARKTKK (SEQ ID NO: 16) of the steroid hormone receptors
(human) glucocorticoid. In general, the one or more NLSs are of
sufficient strength to drive accumulation of the Cas9 in a
detectable amount in the nucleus of a eukaryotic cell. In general,
strength of nuclear localization activity may derive from the
number of NLSs in the Cas9, the particular NLS(s) used, or a
combination of these factors. Detection of accumulation in the
nucleus may be performed by any suitable technique. For example, a
detectable marker may be fused to the Cas9, such that location
within a cell may be visualized, such as in combination with a
means for detecting the location of the nucleus (e.g. a stain
specific for the nucleus such as DAPI). Cell nuclei may also be
isolated from cells, the contents of which may then be analyzed by
any suitable process for detecting protein, such as
immunohistochemistry, Western blot, or enzyme activity assay.
Accumulation in the nucleus may also be determined indirectly, such
as by an assay for the effect of CRISPR complex formation (e.g.
assay for DNA cleavage or mutation at the target sequence, or assay
for altered gene expression activity affected by CRISPR complex
formation and/or Cas enzyme activity), as compared to a control no
exposed to the Cas9 or complex, or exposed to a Cas9 lacking the
one or more NLSs. In other embodiments, no NLS is required.
[0105] In certain aspects the invention involves vectors, e.g. for
delivering or introducing in a cell Cas9 and/or RNA capable of
guiding Cas9 to a target locus (i.e. guide RNA), but also for
propagating these components (e.g. in prokaryotic cells). A used
herein, a "vector" is a tool that allows or facilitates the
transfer of an entity from one environment to another. It is a
replicon, such as a plasmid, phage, or cosmid, into which another
DNA segment may be inserted so as to bring about the replication of
the inserted segment. Generally, a vector is capable of replication
when associated with the proper control elements. In general, the
term "vector" refers to a nucleic acid molecule capable of
transporting another nucleic acid to which it has been linked.
Vectors include, but are not limited to, nucleic acid molecules
that are single-stranded, double-stranded, or partially
double-stranded; nucleic acid molecules that comprise one or more
free ends, no free ends (e.g. circular); nucleic acid molecules
that comprise DNA, RNA, or both; and other varieties of
polynucleotides known in the art. One type of vector is a
"plasmid," which refers to a circular double stranded DNA loop into
which additional DNA segments can be inserted, such as by standard
molecular cloning techniques. Another type of vector is a viral
vector, wherein virally-derived DNA or RNA sequences are present in
the vector for packaging into a virus (e.g. retroviruses,
replication defective retroviruses, adenoviruses, replication
defective adenoviruses, and adeno-associated viruses (AAVs)). Viral
vectors also include polynucleotides carried by a virus for
transfection into a host cell. Certain vectors are capable of
autonomous replication in a host cell into which they are
introduced (e.g. bacterial vectors having a bacterial origin of
replication and episomal mammalian vectors). Other vectors (e.g.,
non-episomal mammalian vectors) are integrated into the genome of a
host cell upon introduction into the host cell, and thereby are
replicated along with the host genome. Moreover, certain vectors
are capable of directing the expression of genes to which they are
operatively-linked. Such vectors are referred to herein as
"expression vectors." Common expression vectors of utility in
recombinant DNA techniques are often in the form of plasmids.
[0106] Recombinant expression vectors can comprise a nucleic acid
of the invention in a form suitable for expression of the nucleic
acid in a host cell, which means that the recombinant expression
vectors include one or more regulatory elements, which may be
selected on the basis of the host cells to be used for expression,
that is operatively-linked to the nucleic acid sequence to be
expressed. Within a recombinant expression vector, "operably
linked" is intended to mean that the nucleotide sequence of
interest is linked to the regulatory element(s) in a manner that
allows for expression of the nucleotide sequence (e.g. in an in
vitro transcription/translation system or in a host cell when the
vector is introduced into the host cell). With regards to
recombination and cloning methods, mention is made of U.S. patent
application Ser. No. 10/815,730, published Sep. 2, 2004 as US
2004-0171156 A1, the contents of which are herein incorporated by
reference in their entirety.
[0107] The vector(s) can include the regulatory element(s), e.g.,
promoter(s). The vector(s) can comprise Cas9 encoding sequences,
and/or a single, but possibly also can comprise at least 3 or 8 or
16 or 32 or 48 or 50 guide RNA(s) (e.g., sgRNAs) encoding
sequences, such as 1-2, 1-3, 1-4 1-5, 3-6, 3-7, 3-8, 3-9, 3-10,
3-8, 3-16, 3-30, 3-32, 3-48, 3-50 RNA(s) (e.g., sgRNAs). In a
single vector there can be a promoter for each RNA (e.g., sgRNA),
advantageously when there are up to about 16 RNA(s) (e.g., sgRNAs);
and, when a single vector provides for more than 16 RNA(s) (e.g.,
sgRNAs), one or more promoter(s) can drive expression of more than
one of the RNA(s) (e.g., sgRNAs), e.g., when there are 32 RNA(s)
(e.g., sgRNAs), each promoter can drive expression of two RNA(s)
(e.g., sgRNAs), and when there are 48 RNA(s) (e.g., sgRNAs), each
promoter can drive expression of three RNA(s) (e.g., sgRNAs). By
simple arithmetic and well established cloning protocols and the
teachings in this disclosure one skilled in the art can readily
practice the invention as to the RNA(s) (e.g., sgRNA(s) for a
suitable exemplary vector such as AAV, and a suitable promoter such
as the U6 promoter, e.g., U6-sgRNAs. For example, the packaging
limit of AAV is .about.4.7 kb. The length of a single U6-sgRNA
(plus restriction sites for cloning) is 361 bp. Therefore, the
skilled person can readily fit about 12-16, e.g., 13 U6-sgRNA
cassettes in a single vector. This can be assembled by any suitable
means, such as a golden gate strategy used for TALE assembly
(http://www.genome-engineering.org/taleffectors/). The skilled
person can also use a tandem guide strategy to increase the number
of U6-sgRNAs by approximately 1.5 times, e.g., to increase from
12-16, e.g., 13 to approximately 18-24, e.g., about 19 U6-sgRNAs.
Therefore, one skilled in the art can readily reach approximately
18-24, e.g., about 19 promoter-RNAs, e.g., U6-sgRNAs in a single
vector, e.g., an AAV vector. A further means for increasing the
number of promoters and RNAs, e.g., sgRNA(s) in a vector is to use
a single promoter (e.g., U6) to express an array of RNAs, e.g.,
sgRNAs separated by cleavable sequences. And an even further means
for increasing the number of promoter-RNAs, e.g., sgRNAs in a
vector, is to express an array of promoter-RNAs, e.g., sgRNAs
separated by cleavable sequences in the intron of a coding sequence
or gene; and, in this instance it is advantageous to use a
polymerase II promoter, which can have increased expression and
enable the transcription of long RNA in a tissue specific manner.
(see, e.g., http://nar.oxfordjournals.org/content/34/7/e53.short,
http://www.nature.com/mt/journal/v16/n9/abs/mt2008144a.html). In an
advantageous embodiment, AAV may package U6 tandem sgRNA targeting
up to about 50 genes. Accordingly, from the knowledge in the art
and the teachings in this disclosure the skilled person can readily
make and use vector(s), e.g., a single vector, expressing multiple
RNAs or guides or sgRNAs under the control or operatively or
functionally linked to one or more promoters--especially as to the
numbers of RNAs or guides or sgRNAs discussed herein, without any
undue experimentation.
[0108] The guide RNA(s), e.g., sgRNA(s) encoding sequences and/or
Cas9 encoding sequences, can be functionally or operatively linked
to regulatory element(s) and hence the regulatory element(s) drive
expression. The promoter(s) can be constitutive promoter(s) and/or
conditional promoter(s) and/or inducible promoter(s) and/or tissue
specific promoter(s). The promoter can be selected from the group
consisting of RNA polymerases, pol I, pol II, pol III, T7, U6, H1,
retroviral Rous sarcoma virus (RSV) LTR promoter, the
cytomegalovirus (CMV) promoter, the SV40 promoter, the
dihydrofolate reductase promoter, the .beta.-actin promoter, the
phosphoglycerol kinase (PGK) promoter, and the EF1.alpha. promoter.
An advantageous promoter is the promoter is U6.
[0109] As used herein, the term "crRNA" or "guide RNA" or "single
guide RNA" or "sgRNA" or "one or more nucleic acid components" of a
Type II CRISPR-Cas9 locus effector protein comprises any
polynucleotide sequence having sufficient complementarity with a
target nucleic acid sequence to hybridize with the target nucleic
acid sequence and direct sequence-specific binding of a nucleic
acid-targeting complex to the target nucleic acid sequence. In some
embodiments, the degree of complementarity, when optimally aligned
using a suitable alignment algorithm, is about or more than about
50%, 60%, 75%, 80%, 85%, 90%, 95%, 97.5%, 99%, or more. Optimal
alignment may be determined with the use of any suitable algorithm
for aligning sequences, non-limiting example of which include the
Smith-Waterman algorithm, the Needleman-Wunsch algorithm,
algorithms based on the Burrows-Wheeler Transform (e.g., the
Burrows Wheeler Aligner), ClustalW, Clustal X, BLAT, Novoalign
(Novocraft Technologies; available at www.novocraft.com), ELAND
(Illumina, San Diego, Calif.), SOAP (available at
soap.genomics.org.cn), and Maq (available at maq.sourceforge.net).
The ability of a guide sequence (within a nucleic acid-targeting
guide RNA) to direct sequence-specific binding of a nucleic
acid-targeting complex to a target nucleic acid sequence may be
assessed by any suitable assay. For example, the components of a
nucleic acid-targeting CRISPR system sufficient to form a nucleic
acid-targeting complex, including the guide sequence to be tested,
may be provided to a host cell having the corresponding target
nucleic acid sequence, such as by transfection with vectors
encoding the components of the nucleic acid-targeting complex,
followed by an assessment of preferential targeting (e.g.,
cleavage) within the target nucleic acid sequence, such as by
Surveyor assay as described herein. Similarly, cleavage of a target
nucleic acid sequence may be evaluated in a test tube by providing
the target nucleic acid sequence, components of a nucleic
acid-targeting complex, including the guide sequence to be tested
and a control guide sequence different from the test guide
sequence, and comparing binding or rate of cleavage at the target
sequence between the test and control guide sequence reactions.
Other assays are possible, and will occur to those skilled in the
art. A guide sequence, and hence a nucleic acid-targeting guide RNA
may be selected to target any target nucleic acid sequence. The
target sequence may be DNA. The target sequence may be any RNA
sequence. In some embodiments, the target sequence may be a
sequence within a RNA molecule selected from the group consisting
of messenger RNA (mRNA), pre-mRNA, ribosomal RNA (rRNA), transfer
RNA (tRNA), micro-RNA (miRNA), small interfering RNA (siRNA), small
nuclear RNA (snRNA), small nucleolar RNA (snoRNA), double stranded
RNA (dsRNA), non coding RNA (ncRNA), long non-coding RNA (lncRNA),
and small cytoplasmatic RNA (scRNA). In some preferred embodiments,
the target sequence may be a sequence within a RNA molecule
selected from the group consisting of mRNA, pre-mRNA, and rRNA. In
some preferred embodiments, the target sequence may be a sequence
within a RNA molecule selected from the group consisting of ncRNA,
and lncRNA. In some more preferred embodiments, the target sequence
may be a sequence within an mRNA molecule or a pre-mRNA
molecule.
[0110] In some embodiments, a nucleic acid-targeting guide RNA is
selected to reduce the degree secondary structure within the
RNA-targeting guide RNA. In some embodiments, about or less than
about 75%, 50%, 40%, 30%, 25%, 20%, 15%, 10%, 5%, 1%, or fewer of
the nucleotides of the nucleic acid-targeting guide RNA participate
in self-complementary base pairing when optimally folded. Optimal
folding may be determined by any suitable polynucleotide folding
algorithm. Some programs are based on calculating the minimal Gibbs
free energy. An example of one such algorithm is mFold, as
described by Zuker and Stiegler (Nucleic Acids Res. 9 (1981),
133-148). Another example folding algorithm is the online webserver
RNAfold, developed at Institute for Theoretical Chemistry at the
University of Vienna, using the centroid structure prediction
algorithm (see e.g., A. R. Gruber et al., 2008, Cell 106(1): 23-24;
and PA Carr and GM Church, 2009, Nature Biotechnology 27(12):
1151-62).
[0111] In certain embodiments, a guide RNA or crRNA may comprise,
consist essentially of, or consist of a direct repeat (DR) sequence
and a guide sequence or spacer sequence. In certain embodiments,
the guide RNA or crRNA may comprise, consist essentially of, or
consist of a direct repeat sequence fused or linked to a guide
sequence or spacer sequence. In certain embodiments, the direct
repeat sequence may be located upstream (i.e., 5') from the guide
sequence or spacer sequence. In other embodiments, the direct
repeat sequence may be located downstream (i.e., 3') from the guide
sequence or spacer sequence.
[0112] In certain embodiments, the crRNA comprises a stem loop,
preferably a single stem loop. In certain embodiments, the direct
repeat sequence forms a stem loop, preferably a single stem
loop.
[0113] A guide sequence may be selected to target any target
sequence. In some embodiments, the target sequence is a sequence
within a genome of a cell. Exemplary target sequences include those
that are unique in the target genome. For example, for the S.
pyogenes Cas9, a unique target sequence in a genome may include a
Cas9 target site of the form MMMMMMMMNNNNNNNNNNNNXGG (SEQ ID NO:
62) where NNNNNNNNNNNNXGG (SEQ ID NO: 63) (N is A, G, T, or C; and
X can be anything) has a single occurrence in the genome. A unique
target sequence in a genome may include an S. pyogenes Cas9 target
site of the form MMMMMMMMMNNNNNNNNNNNXGG (SEQ ID NO: 64) where
NNNNNNNNNNNXGG (SEQ ID NO: 65) (N is A, G, T, or C; and X can be
anything) has a single occurrence in the genome. For the S.
thermophilus CRISPR1 Cas9, a unique target sequence in a genome may
include a Cas9 target site of the form MMMMMMMMNNNNNNNNNNNNXXAGAAW
(SEQ ID NO: 17) where NNNNNNNNNNNNXXAGAAW (SEQ ID NO: 18) (N is A,
G, T, or C; X can be anything; and W is A or T) has a single
occurrence in the genome. A unique target sequence in a genome may
include an S. thermophilus CRISPR1 Cas9 target site of the form
MMMMMMMMMNNNNNNNNNNNXXAGAAW (SEQ ID NO: 19) where
NNNNNNNNNNNXXAGAAW (SEQ ID NO: 20) (N is A, G, T, or C; X can be
anything; and W is A or T) has a single occurrence in the genome.
For the S. pyogenes Cas9, a unique target sequence in a genome may
include a Cas9 target site of the form MMMMMMMMNNNNNNNNNNNNXGGXG
(SEQ ID NO: 66) where NNNNNNNNNNNNXGGXG (SEQ ID NO: 67) (N is A, G,
T, or C; and X can be anything) has a single occurrence in the
genome. A unique target sequence in a genome may include an S.
pyogenes Cas9 target site of the form MMMMMMMMMNNNNNNNNNNNXGGXG
(SEQ ID NO: 68) where NNNNNNNNNNNXGGXG (SEQ ID NO: 69) (N is A, G,
T, or C; and X can be anything) has a single occurrence in the
genome. In each of these sequences "M" may be A, G, T, or C, and
need not be considered in identifying a sequence as unique. In some
embodiments, a guide sequence is selected to reduce the degree
secondary structure within the guide sequence. In some embodiments,
about or less than about 75%, 50%, 40%, 30%, 25%, 20%, 15%, 10%,
5%, 1%, or fewer of the nucleotides of the guide sequence
participate in self-complementary base pairing when optimally
folded. Optimal folding may be determined by any suitable
polynucleotide folding algorithm. Some programs are based on
calculating the minimal Gibbs free energy. An example of one such
algorithm is mFold, as described by Zuker and Stiegler (Nucleic
Acids Res. 9 (1981), 133-148). Another example folding algorithm is
the online webserver RNAfold, developed at Institute for
Theoretical Chemistry at the University of Vienna, using the
centroid structure prediction algorithm (see e.g., A. R. Gruber et
al., 2008, Cell 106(1): 23-24; and PA Carr and GM Church, 2009,
Nature Biotechnology 27(12): 1151-62).
[0114] In general, a tracr mate sequence includes any sequence that
has sufficient complementarity with a tracr sequence to promote one
or more of: (1) excision of a guide sequence flanked by tracr mate
sequences in a cell containing the corresponding tracr sequence;
and (2) formation of a CRISPR complex at a target sequence, wherein
the CRISPR complex comprises the tracr mate sequence hybridized to
the tracr sequence. In general, degree of complementarity is with
reference to the optimal alignment of the tracr mate sequence and
tracr sequence, along the length of the shorter of the two
sequences. Optimal alignment may be determined by any suitable
alignment algorithm, and may further account for secondary
structures, such as self-complementarity within either the tracr
sequence or tracr mate sequence. In some embodiments, the degree of
complementarity between the tracr sequence and tracr mate sequence
along the length of the shorter of the two when optimally aligned
is about or more than about 25%, 30%, 40%, 50%, 60%, 70%, 80%, 90%,
95%, 97.5%, 99%, or higher. In some embodiments, the tracr sequence
is about or more than about 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15,
16, 17, 18, 19, 20, 25, 30, 40, 50, or more nucleotides in length.
In some embodiments, the tracr sequence and tracr mate sequence are
contained within a single transcript, such that hybridization
between the two produces a transcript having a secondary structure,
such as a hairpin. In an embodiment of the invention, the
transcript or transcribed polynucleotide sequence has at least two
or more hairpins. In preferred embodiments, the transcript has two,
three, four or five hairpins. In a further embodiment of the
invention, the transcript has at most five hairpins. In a hairpin
structure the portion of the sequence 5' of the final "N" and
upstream of the loop corresponds to the tracr mate sequence, and
the portion of the sequence 3' of the loop corresponds to the tracr
sequence Further non-limiting examples of single polynucleotides
comprising a guide sequence, a tracr mate sequence, and a tracr
sequence are as follows (listed 5' to 3'), where "N" represents a
base of a guide sequence, the first block of lower case letters
represent the tracr mate sequence, and the second block of lower
case letters represent the tracr sequence, and the final poly-T
sequence represents the transcription terminator: (1)
NNNNNNNNNNNNNNNNNNNNgtttttgtactctcaagatttaGAAAtaaatcttgcagaagctacaaagataa
ggcttcatgccgaaatcaacaccctgtcattttatggcagggtgttttcgttatttaaTTTTTT
(SEQ ID NO: 21); (2)
NNNNNNNNNNNNNNNNNNNNgtttttgtactctcaGAAAtgcagaagctacaaagataaggcttcatgccg
aaatcaacaccctgtcattttatggcagggtgttttcgttatttaaTTTTTT (SEQ ID NO:
22); (3)
NNNNNNNNNNNNNNNNNNNNgtttttgtactctcaGAAAtgcagaagctacaaagataaggcttcatgccg
aaatcaacaccctgtcattttatggcagggtgtTTTTTT (SEQ ID NO: 23); (4)
NNNNNNNNNNNNNNNNNNNNgttttagagctaGAAAtagcaagttaaaataaggctagtccgttatcaactt
gaaaaagtggcaccgagtcggtgcTTTTTT (SEQ ID NO: 24); (5)
NNNNNNNNNNNNNNNNNNNNgttttagagctaGAAATAGcaagttaaaataaggctagtccgttatcaac
ttgaaaaagtgTTTTTTT (SEQ ID NO: 25); and (6)
NNNNNNNNNNNNNNNNNNNNgttttagagctagAAATAGcaagttaaaataaggctagtccgttatcaTT
TTTTTT (SEQ ID NO: 26). In some embodiments, sequences (1) to (3)
are used in combination with Cas9 from S. thermophilus CRISPR1. In
some embodiments, sequences (4) to (6) are used in combination with
Cas9 from S. pyogenes. In some embodiments, the tracr sequence is a
separate transcript from a transcript comprising the tracr mate
sequence.
[0115] In some embodiments, candidate tracrRNA may be subsequently
predicted by sequences that fulfill any or all of the following
criteria: 1. sequence homology to direct repeats (motif search in
Geneious with up to 18-bp mismatches); 2. presence of a predicted
Rho-independent transcriptional terminator in direction of
transcription; and 3. stable hairpin secondary structure between
tracrRNA and direct repeat. In some embodiments, 2 of these
criteria may be used, for instance 1 and 2, 2 and 3, or 1 and 3. In
some embodiments, all 3 criteria may be used.
[0116] In some embodiments, chimeric synthetic guide RNAs (sgRNAs)
designs may incorporate at least 12 bp of duplex structure between
the direct repeat and tracrRNA.
[0117] For minimization of toxicity and off-target effect, it will
be important to control the concentration of CRISPR enzyme mRNA and
guide RNA delivered. Optimal concentrations of CRISPR enzyme mRNA
and guide RNA can be determined by testing different concentrations
in a cellular or non-human eukaryote animal model and using deep
sequencing the analyze the extent of modification at potential
off-target genomic loci. For example, for the guide sequence
targeting 5'-GAGTCCGAGCAGAAGAAGAA-3' (SEQ ID NO: 45) in the EMX1
gene of the human genome, deep sequencing can be used to assess the
level of modification at the following two off-target loci, 1:
5'-GAGTCCTAGCAGGAGAAGAA-3' (SEQ ID NO: 46) and 2:
5'-GAGTCTAAGCAGAAGAAGAA-3' (SEQ ID NO: 47). The concentration that
gives the highest level of on-target modification while minimizing
the level of off-target modification should be chosen for in vivo
delivery. Alternatively, to minimize the level of toxicity and
off-target effect, CRISPR enzyme nickase mRNA (for example S.
pyogenes Cas9 with the D10A mutation) can be delivered with a pair
of guide RNAs targeting a site of interest. The two guide RNAs need
to be spaced as follows. Guide sequences and strategies to minimize
toxicity and off-target effects can be as in WO 2014/093622
(PCT/US2013/074667).
[0118] The term "nucleic acid-targeting system", wherein nucleic
acid is DNA or RNA, and in some aspects may also refer to DNA-RNA
hybrids or derivatives thereof, refers collectively to transcripts
and other elements involved in the expression of or directing the
activity of DNA or RNA-targeting CRISPR-associated ("Cas9") genes,
which may include sequences encoding a DNA or RNA-targeting Cas9
protein and a DNA or RNA-targeting guide RNA comprising a CRISPR
RNA (crRNA) sequence and (in some but not all systems) a
trans-activating CRISPR/Cas9 system RNA (tracrRNA) sequence, or
other sequences and transcripts from a DNA or RNA-targeting CRISPR
locus. In general, a RNA-targeting system is characterized by
elements that promote the formation of a DNA or RNA-targeting
complex at the site of a target DNA or RNA sequence. In the context
of formation of a DNA or RNA-targeting complex, "target sequence"
refers to a DNA or RNA sequence to which a DNA or RNA-targeting
guide RNA is designed to have complementarity, where hybridization
between a target sequence and a RNA-targeting guide RNA promotes
the formation of a RNA-targeting complex. In some embodiments, a
target sequence is located in the nucleus or cytoplasm of a
cell.
[0119] In an aspect of the invention, novel DNA targeting systems
also referred to as DNA-targeting CRISPR/Cas9 or the CRISPR-Cas9
DNA-targeting system of the present application are based on
identified Type II Cas9 proteins which do not require the
generation of customized proteins to target specific DNA sequences
but rather a single effector protein or enzyme can be programmed by
a RNA molecule to recognize a specific DNA target, in other words
the enzyme can be recruited to a specific DNA target using said RNA
molecule. The invention particularly relates to DNA targeting
RNA-guided Cas9 CRISPR systems.
[0120] In an aspect of the invention, novel RNA targeting systems
also referred to as RNA- or RNA-targeting CRISPR/Cas9 or the
CRISPR-Cas9 system RNA-targeting system of the present application
are based on identified Type II Cas9 proteins which do not require
the generation of customized proteins to target specific RNA
sequences but rather a single enzyme can be programmed by a RNA
molecule to recognize a specific RNA target, in other words the
enzyme can be recruited to a specific RNA target using said RNA
molecule.
[0121] The nucleic acids-targeting systems, the vector systems, the
vectors and the compositions described herein may be used in
various nucleic acids-targeting applications, altering or modifying
synthesis of a gene product, such as a protein, nucleic acids
cleavage, nucleic acids editing, nucleic acids splicing;
trafficking of target nucleic acids, tracing of target nucleic
acids, isolation of target nucleic acids, visualization of target
nucleic acids, etc.
[0122] Aspects of the invention also encompass methods and uses of
the compositions and systems described herein in genome
engineering, e.g. for altering or manipulating the expression of
one or more genes or the one or more gene products, in prokaryotic
or eukaryotic cells, in vitro, in vivo or ex vivo.
[0123] The CRISPR system is derived advantageously from a type II
CRISPR system. In some embodiments, one or more elements of a
CRISPR system is derived from a particular organism comprising an
endogenous CRISPR system, such as Streptococcus pyogenes. The
CRISPR system is a type II CRISPR system and the Cas enzyme is
Cas9, which catalyzes DNA cleavage. Non-limiting examples of Cas
proteins include Cas1, Cas1B, Cas2, Cas3, Cas4, Cas5, Cas6, Cas7,
Cas8, Cas9 (also known as Csn1 and Csx12), Cas10, Csy1, Csy2, Csy3,
Cse1, Cse2, Csc1, Csc2, Csa5, Csn2, Csm2, Csm3, Csm4, Csm5, Csm6,
Cmr1, Cmr3, Cmr4, Cmr5, Cmr6, Csb1, Csb2, Csb3, Csx17, Csx14,
Csx10, Csx16, CsaX, Csx3, Csx1, Csx15, Csf1, Csf2, Csf3, Csf4,
homologues thereof, or modified versions thereof.
[0124] In an embodiment, the Cas9 protein may be an ortholog of an
organism of a genus which includes but is not limited to
Corynebacter, Sutterella, Legionella, Treponema, Filifactor,
Eubacterium, Streptococcus, Lactobacillus, Mycoplasma, Bacteroides,
Flaviivola, Flavobacterium, Sphaerochaeta, Azospirillum,
Gluconacetobacter, Neisseria, Roseburia, Parvibaculum,
Staphylococcus, Nitratifractor, Mycoplasma and Campylobacter.
Species of an organism of such a genus can be as otherwise herein
discussed.
[0125] Some methods of identifying orthologs of CRISPR-Cas system
enzymes may involve identifying tracr sequences in genomes of
interest. Identification of tracr sequences may relate to the
following steps: Search for the direct repeats or tracr mate
sequences in a database to identify a CRISPR region comprising a
CRISPR enzyme. Search for homologous sequences in the CRISPR region
flanking the CRISPR enzyme in both the sense and antisense
directions. Look for transcriptional terminators and secondary
structures. Identify any sequence that is not a direct repeat or a
tracr mate sequence but has more than 50% identity to the direct
repeat or tracr mate sequence as a potential tracr sequence. Take
the potential tracr sequence and analyze for transcriptional
terminator sequences associated therewith.
[0126] It will be appreciated that any of the functionalities
described herein may be engineered into CRISPR enzymes from other
orthologs, including chimeric enzymes comprising fragments from
multiple orthologs. Examples of such orthologs are described
elsewhere herein. Thus, chimeric enzymes may comprise fragments of
CRISPR enzyme orthologs of an organism which includes but is not
limited to Corynebacter, Sutterella, Legionella, Treponema,
Filifactor, Eubacterium, Streptococcus, Lactobacillus, Mycoplasma,
Bacteroides, Flaviivola, Flavobacterium, Sphaerochaeta,
Azospirillum, Gluconacetobacter, Neisseria, Roseburia,
Parvibaculum, Staphylococcus, Nitratifractor, Mycoplasma and
Campylobacter. A chimeric enzyme can comprise a first fragment and
a second fragment, and the fragments can be of CRISPR enzyme
orthologs of organisms of genuses herein mentioned or of species
herein mentioned; advantageously the fragments are from CRISPR
enzyme orthologs of different species
[0127] In some embodiments, the unmodified CRISPR Cas 9 enzyme has
DNA cleavage activity. In some embodiments, the CRISPR enzyme
directs cleavage of one or both strands at the location of a target
sequence, such as within the target sequence and/or within the
complement of the target sequence. In some embodiments, the CRISPR
enzyme directs cleavage of one or both strands within about 1, 2,
3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 50, 100, 200, 500, or more
base pairs from the first or last nucleotide of a target sequence.
In some embodiments, a vector encodes a CRISPR enzyme that is
mutated to with respect to a corresponding wild-type enzyme such
that the mutated CRISPR enzyme lacks the ability to cleave one or
both strands of a target polynucleotide containing a target
sequence. For example, an aspartate-to-alanine substitution (D10A)
in the RuvC I catalytic domain of Cas9 from S. pyogenes converts
Cas9 from a nuclease that cleaves both strands to a nickase
(cleaves a single strand). Other examples of mutations that render
Cas9 a nickase include, without limitation, H840A, N854A, and
N863A. As a further example, two or more catalytic domains of Cas9
(RuvC I, RuvC II, and RuvC III or the HNH domain) may be mutated to
produce a mutated Cas9 substantially lacking all DNA cleavage
activity. In some embodiments, a D10A mutation is combined with one
or more of H840A, N854A, or N863A mutations to produce a Cas9
enzyme substantially lacking all DNA cleavage activity. In some
embodiments, a CRISPR enzyme is considered to substantially lack
all DNA cleavage activity when the DNA cleavage activity of the
mutated enzyme is about no more than 25%, 10%, 5%, 1%, 0.1%, 0.01%,
or less of the DNA cleavage activity of the non-mutated form of the
enzyme; an example can be when the DNA cleavage activity of the
mutated form is nil or negligible as compared with the non-mutated
form. Where the enzyme is not SpCas9, mutations may be made at any
or all residues corresponding to positions 10, 762, 840, 854, 863
and/or 986 of SpCas9 (which may be ascertained for instance by
standard sequence comparison tools). In particular, any or all of
the following mutations are preferred in SpCas9: D10A, E762A,
H840A, N854A, N863A and/or D986A; as well as conservative
substitution for any of the replacement amino acids is also
envisaged. The same (or conservative substitutions of these
mutations) at corresponding positions in other Cas9s are also
preferred. Particularly preferred are D10 and H840 in SpCas9.
However, in other Cas9s, residues corresponding to SpCas9 D10 and
H840 are also preferred. Orthologs of SpCas9 can be used in the
practice of the invention. A Cas enzyme may be identified Cas9 as
this can refer to the general class of enzymes that share homology
to the biggest nuclease with multiple nuclease domains from the
type II CRISPR system. Most preferably, the Cas9 enzyme is from, or
is derived from, spCas9 (S. pyogenes Cas9) or saCas9 (S. aureus
Cas9). StCas9'' refers to wild type Cas9 from S. thermophilus, the
protein sequence of which is given in the SwissProt database under
accession number G3ECR1. Similarly, S pyogenes Cas9 or spCas9 is
included in SwissProt under accession number Q99ZW2. By derived,
Applicants mean that the derived enzyme is largely based, in the
sense of having a high degree of sequence homology with, a wildtype
enzyme, but that it has been mutated (modified) in some way as
described herein. It will be appreciated that the terms Cas and
CRISPR enzyme are generally used herein interchangeably, unless
otherwise apparent. As mentioned above, many of the residue
numberings used herein refer to the Cas9 enzyme from the type II
CRISPR locus in Streptococcus pyogenes. However, it will be
appreciated that this invention includes many more Cas9s from other
species of microbes, such as SpCas9, SaCa9, St1Cas9 and so forth.
Enzymatic action by Cas9 derived from Streptococcus pyogenes or any
closely related Cas9 generates double stranded breaks at target
site sequences which hybridize to 20 nucleotides of the guide
sequence and that have a protospacer-adjacent motif (PAM) sequence
(examples include NGG/NRG or a PAM that can be determined as
described herein) following the 20 nucleotides of the target
sequence. CRISPR activity through Cas9 for site-specific DNA
recognition and cleavage is defined by the guide sequence, the
tracr sequence that hybridizes in part to the guide sequence and
the PAM sequence. More aspects of the CRISPR system are described
in Karginov and Hannon, The CRISPR system: small RNA-guided defense
in bacteria and archaea, Mole Cell 2010, January 15; 37(1): 7. The
type II CRISPR locus from Streptococcus pyogenes SF370, which
contains a cluster of four genes Cas9, Cas1, Cas2, and Csn1, as
well as two non-coding RNA elements, tracrRNA and a characteristic
array of repetitive sequences (direct repeats) interspaced by short
stretches of non-repetitive sequences (spacers, about 30 bp each).
In this system, targeted DNA double-strand break (DSB) is generated
in four sequential steps. First, two non-coding RNAs, the pre-crRNA
array and tracrRNA, are transcribed from the CRISPR locus. Second,
tracrRNA hybridizes to the direct repeats of pre-crRNA, which is
then processed into mature crRNAs containing individual spacer
sequences. Third, the mature crRNA:tracrRNA complex directs Cas9 to
the DNA target comprising, consisting essentially of, or consisting
of the protospacer and the corresponding PAM via heteroduplex
formation between the spacer region of the crRNA and the
protospacer DNA. Finally, Cas9 mediates cleavage of target DNA
upstream of PAM to create a DSB within the protospacer. A pre-crRNA
array comprising, consisting essentially of, or consisting of a
single spacer flanked by two direct repeats (DRs) is also
encompassed by the term "tracr-mate sequences"). In certain
embodiments, Cas9 may be constitutively present or inducibly
present or conditionally present or administered or delivered. Cas9
optimization may be used to enhance function or to develop new
functions, one can generate chimeric Cas9 proteins. And Cas9 may be
used as a generic DNA binding protein.
[0128] Typically, in the context of an endogenous CRISPR system,
formation of a CRISPR complex (comprising a guide sequence
hybridized to a target sequence and complexed with one or more Cas9
proteins) results in cleavage of one or both strands in or near
(e.g., within 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 50, or more base
pairs from) the target sequence. Without wishing to be bound by
theory, the tracr sequence, which may comprise, consist essentially
of, or consist of all or a portion of a wild-type tracr sequence
(e.g., about or more than about 20, 26, 32, 45, 48, 54, 63, 67, 85,
or more nucleotides of a wild-type tracr sequence), may also form
part of a CRISPR complex, such as by hybridization along at least a
portion of the tracr sequence to all or a portion of a tracr mate
sequence that is operably linked to the guide sequence.
[0129] An example of a codon optimized sequence, is in this
instance a sequence optimized for expression in a eukaryote, e.g.,
humans (i.e. being optimized for expression in humans), or for
another eukaryote, animal or mammal as herein discussed; see, e.g.,
SaCas9 human codon optimized sequence in WO 2014/093622
(PCT/US2013/074667). While this is preferred, it will be
appreciated that other examples are possible and codon optimization
for a host species other than human, or for codon optimization for
specific organs is known. In some embodiments, a coding sequence
encoding a CRISPR enzyme is codon optimized for expression in
particular cells, such as eukaryotic cells. The eukaryotic cells
may be those of or derived from a particular organism, such as a
mammal, including but not limited to human, or non-human eukaryote
or animal or mammal as herein discussed, e.g., mouse, rat, rabbit,
dog, livestock, or non-human mammal or primate. In some
embodiments, processes for modifying the germ line genetic identity
of human beings and/or processes for modifying the genetic identity
of animals which are likely to cause them suffering without any
substantial medical benefit to man or animal, and also animals
resulting from such processes, may be excluded. In general, codon
optimization refers to a process of modifying a nucleic acid
sequence for enhanced expression in the host cells of interest by
replacing at least one codon (e.g., about or more than about 1, 2,
3, 4, 5, 10, 15, 20, 25, 50, or more codons) of the native sequence
with codons that are more frequently or most frequently used in the
genes of that host cell while maintaining the native amino acid
sequence. Various species exhibit particular bias for certain
codons of a particular amino acid. Codon bias (differences in codon
usage between organisms) often correlates with the efficiency of
translation of messenger RNA (mRNA), which is in turn believed to
be dependent on, among other things, the properties of the codons
being translated and the availability of particular transfer RNA
(tRNA) molecules. The predominance of selected tRNAs in a cell is
generally a reflection of the codons used most frequently in
peptide synthesis. Accordingly, genes can be tailored for optimal
gene expression in a given organism based on codon optimization.
Codon usage tables are readily available, for example, at the
"Codon Usage Database" available at www.kazusa.orjp/codon/ and
these tables can be adapted in a number of ways. See Nakamura, Y.,
et al. "Codon usage tabulated from the international DNA sequence
databases: status for the year 2000" Nucl. Acids Res. 28:292
(2000). Computer algorithms for codon optimizing a particular
sequence for expression in a particular host cell are also
available, such as Gene Forge (Aptagen; Jacobus, Pa.), are also
available. In some embodiments, one or more codons (e.g., 1, 2, 3,
4, 5, 10, 15, 20, 25, 50, or more, or all codons) in a sequence
encoding a CRISPR enzyme correspond to the most frequently used
codon for a particular amino acid.
[0130] In some embodiments, a vector encodes a CRISPR enzyme
comprising one or more nuclear localization sequences (NLSs), such
as about or more than about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more
NLSs. In some embodiments, the CRISPR enzyme comprises about or
more than about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more NLSs at or
near the amino-terminus, about or more than about 1, 2, 3, 4, 5, 6,
7, 8, 9, 10, or more NLSs at or near the carboxy-terminus, or a
combination of these (e.g., zero or at least one or more NLS at the
amino-terminus and zero or at one or more NLS at the carboxy
terminus). When more than one NLS is present, each may be selected
independently of the others, such that a single NLS may be present
in more than one copy and/or in combination with one or more other
NLSs present in one or more copies. In a preferred embodiment of
the invention, the CRISPR enzyme comprises at most 6 NLSs. In some
embodiments, an NLS is considered near the N- or C-terminus when
the nearest amino acid of the NLS is within about 1, 2, 3, 4, 5,
10, 15, 20, 25, 30, 40, 50, or more amino acids along the
polypeptide chain from the N- or C-terminus. Non-limiting examples
of NLSs include an NLS sequence derived from: the NLS of the SV40
virus large T-antigen, having the amino acid sequence PKKKRKV (SEQ
ID NO: 1); the NLS from nucleoplasmin (e.g., the nucleoplasmin
bipartite NLS with the sequence KRPAATKKAGQAKKKK (SEQ ID NO: 2));
the c-myc NLS having the amino acid sequence PAAKRVKLD (SEQ ID NO:
3) or RQRRNELKRSP (SEQ ID NO: 4); the hRNPA1 M9 NLS having the
sequence NQSSNFGPMKGGNFGGRSSGPYGGGGQYFAKPRNQGGY (SEQ ID NO: 5); the
sequence RMRIZFKNKGKDTAELRRRRVEVSVELRKAKKDEQILKRRNV (SEQ ID NO: 6)
of the IBB domain from importin-alpha; the sequences VSRKRPRP (SEQ
ID NO: 7) and PPKKARED (SEQ ID NO: 8) of the myoma T protein; the
sequence PQPKKKPL (SEQ ID NO: 9) of human p53; the sequence
SALIKKKKKMAP (SEQ ID NO: 10) of mouse c-abl IV; the sequences DRLRR
(SEQ ID NO: 11) and PKQKKRK (SEQ ID NO: 12) of the influenza virus
NS1; the sequence RKLKKKIKKL (SEQ ID NO: 13) of the Hepatitis virus
delta antigen; the sequence REKKKFLKRR (SEQ ID NO: 14) of the mouse
Mx1 protein; the sequence KRKGDEVDGVDEVAKKKSKK (SEQ ID NO: 15) of
the human poly(ADP-ribose) polymerase; and the sequence
RKCLQAGMNLEARKTKK (SEQ ID NO: 16) of the steroid hormone receptors
(human) glucocorticoid. In general, the one or more NLSs are of
sufficient strength to drive accumulation of the CRISPR enzyme in a
detectable amount in the nucleus of a eukaryotic cell. In general,
strength of nuclear localization activity may derive from the
number of NLSs in the CRISPR enzyme, the particular NLS(s) used, or
a combination of these factors. Detection of accumulation in the
nucleus may be performed by any suitable technique. For example, a
detectable marker may be fused to the CRISPR enzyme, such that
location within a cell may be visualized, such as in combination
with a means for detecting the location of the nucleus (e.g., a
stain specific for the nucleus such as DAPI). Cell nuclei may also
be isolated from cells, the contents of which may then be analyzed
by any suitable process for detecting protein, such as
immunohistochemistry, Western blot, or enzyme activity assay.
Accumulation in the nucleus may also be determined indirectly, such
as by an assay for the effect of CRISPR complex formation (e.g.,
assay for DNA cleavage or mutation at the target sequence, or assay
for altered gene expression activity affected by CRISPR complex
formation and/or CRISPR enzyme activity), as compared to a control
no exposed to the CRISPR enzyme or complex, or exposed to a CRISPR
enzyme lacking the one or more NLSs.
[0131] Aspects of the invention relate to the expression of the
gene product being decreased or a template polynucleotide being
further introduced into the DNA molecule encoding the gene product
or an intervening sequence being excised precisely by allowing the
two 5' overhangs to reanneal and ligate or the activity or function
of the gene product being altered or the expression of the gene
product being increased. In an embodiment of the invention, the
gene product is a protein. Only sgRNA pairs creating 5' overhangs
with less than 8 bp overlap between the guide sequences (offset
greater than .about.8 bp) were able to mediate detectable indel
formation. Importantly, each guide used in these assays is able to
efficiently induce indels when paired with wildtype Cas9,
indicating that the relative positions of the guide pairs are the
most important parameters in predicting double nicking activity.
Since Cas9n and Cas9H840A nick opposite strands of DNA,
substitution of Cas9n with Cas9H840A with a given sgRNA pair should
have resulted in the inversion of the overhang type; but no indel
formation is observed as with Cas9H840A indicating that Cas9H840A
is a CRISPR enzyme substantially lacking all DNA cleavage activity
(which is when the DNA cleavage activity of the mutated enzyme is
about no more than 25%, 10%, 5%, 1%, 0.1%, 0.01%, or less of the
DNA cleavage activity of the non-mutated form of the enzyme;
whereby an example can be when the DNA cleavage activity of the
mutated form is nil or negligible as compared with the non-mutated
form, e.g., when no indel formation is observed as with Cas9H840A
in the eukaryotic system in contrast to the biochemical or
prokaryotic systems). Nonetheless, a pair of sgRNAs that will
generate a 5' overhang with Cas9n should in principle generate the
corresponding 3' overhang instead, and double nicking. Therefore,
sgRNA pairs that lead to the generation of a 3' overhang with Cas9n
can be used with another mutated Cas9 to generate a 5' overhang,
and double nicking. Accordingly, in some embodiments, a
recombination template is also provided. A recombination template
may be a component of another vector as described herein, contained
in a separate vector, or provided as a separate polynucleotide. In
some embodiments, a recombination template is designed to serve as
a template in homologous recombination, such as within or near a
target sequence nicked or cleaved by a CRISPR enzyme as a part of a
CRISPR complex. A template polynucleotide may be of any suitable
length, such as about or more than about 10, 15, 20, 25, 50, 75,
100, 150, 200, 500, 1000, or more nucleotides in length. In some
embodiments, the template polynucleotide is complementary to a
portion of a polynucleotide comprising the target sequence. When
optimally aligned, a template polynucleotide might overlap with one
or more nucleotides of a target sequences (e.g., about or more than
about 1, 5, 10, 15, 20, or more nucleotides). In some embodiments,
when a template sequence and a polynucleotide comprising a target
sequence are optimally aligned, the nearest nucleotide of the
template polynucleotide is within about 1, 5, 10, 15, 20, 25, 50,
75, 100, 200, 300, 400, 500, 1000, 5000, 10000, or more nucleotides
from the target sequence.
[0132] In some embodiments, one or more vectors driving expression
of one or more elements of a CRISPR system are introduced into a
host cell such that expression of the elements of the CRISPR system
direct formation of a CRISPR complex at one or more target sites.
For example, a Cas enzyme, a guide sequence linked to a tracr-mate
sequence, and a tracr sequence could each be operably linked to
separate regulatory elements on separate vectors. Or, RNA(s) of the
CRISPR System can be delivered to a transgenic Cas9 animal or
mammal, e.g., an animal or mammal that constitutively or inducibly
or conditionally expresses Cas9; or an animal or mammal that is
otherwise expressing Cas9 or has cells containing Cas9, such as by
way of prior administration thereto of a vector or vectors that
code for and express in vivo Cas9. Alternatively, two or more of
the elements expressed from the same or different regulatory
elements, may be combined in a single vector, with one or more
additional vectors providing any components of the CRISPR system
not included in the first vector. CRISPR system elements that are
combined in a single vector may be arranged in any suitable
orientation, such as one element located 5' with respect to
("upstream" of) or 3' with respect to ("downstream" of) a second
element. The coding sequence of one element may be located on the
same or opposite strand of the coding sequence of a second element,
and oriented in the same or opposite direction. In some
embodiments, a single promoter drives expression of a transcript
encoding a CRISPR enzyme and one or more of the guide sequence,
tracr mate sequence (optionally operably linked to the guide
sequence), and a tracr sequence embedded within one or more intron
sequences (e.g., each in a different intron, two or more in at
least one intron, or all in a single intron). In some embodiments,
the CRISPR enzyme, guide sequence, tracr mate sequence, and tracr
sequence are operably linked to and expressed from the same
promoter. Delivery vehicles, vectors, particles, nanoparticles,
formulations and components thereof for expression of one or more
elements of a CRISPR system are as used in the foregoing documents,
such as WO 2014/093622 (PCT/US2013/074667). In some embodiments, a
vector comprises one or more insertion sites, such as a restriction
endonuclease recognition sequence (also referred to as a "cloning
site"). In some embodiments, one or more insertion sites (e.g.,
about or more than about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more
insertion sites) are located upstream and/or downstream of one or
more sequence elements of one or more vectors. In some embodiments,
a vector comprises an insertion site upstream of a tracr mate
sequence, and optionally downstream of a regulatory element
operably linked to the tracr mate sequence, such that following
insertion of a guide sequence into the insertion site and upon
expression the guide sequence directs sequence-specific binding of
a CRISPR complex to a target sequence in a eukaryotic cell. In some
embodiments, a vector comprises two or more insertion sites, each
insertion site being located between two tracr mate sequences so as
to allow insertion of a guide sequence at each site. In such an
arrangement, the two or more guide sequences may comprise two or
more copies of a single guide sequence, two or more different guide
sequences, or combinations of these. When multiple different guide
sequences are used, a single expression construct may be used to
target CRISPR activity to multiple different, corresponding target
sequences within a cell. For example, a single vector may comprise
about or more than about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, or
more guide sequences. In some embodiments, about or more than about
1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more such
guide-sequence-containing vectors may be provided, and optionally
delivered to a cell. In some embodiments, a vector comprises a
regulatory element operably linked to an enzyme-coding sequence
encoding a CRISPR enzymeCas9 protein. CRISPR enzyme or CRISPR
enzyme mRNA or CRISPR guide RNA or RNA(s) can be delivered
separately; and advantageously at least one of these is delivered
via a nanoparticle complex. CRISPR enzyme mRNA can be delivered
prior to the guide RNA to give time for CRISPR enzyme to be
expressed. CRISPR enzyme mRNA might be administered 1-12 hours
(preferably around 2-6 hours) prior to the administration of guide
RNA. Alternatively, CRISPR enzyme mRNA and guide RNA can be
administered together. Advantageously, a second booster dose of
guide RNA can be administered 1-12 hours (preferably around 2-6
hours) after the initial administration of CRISPR enzyme mRNA+guide
RNA. Additional administrations of CRISPR enzyme mRNA and/or guide
RNA might be useful to achieve the most efficient levels of genome
modification.
[0133] In one aspect, the invention provides methods for using one
or more elements of a CRISPR system. The CRISPR complex of the
invention provides an effective means for modifying a target
polynucleotide. The CRISPR complex of the invention has a wide
variety of utility including modifying (e.g., deleting, inserting,
translocating, inactivating, activating) a target polynucleotide in
a multiplicity of cell types. As such the CRISPR complex of the
invention has a broad spectrum of applications in, e.g., gene
therapy, drug screening, disease diagnosis, and prognosis. An
exemplary CRISPR complex comprises a CRISPR enzyme complexed with a
guide sequence hybridized to a target sequence within the target
polynucleotide. The guide sequence is linked to a tracr mate
sequence, which in turn hybridizes to a tracr sequence. In one
embodiment, this invention provides a method of cleaving a target
polynucleotide. The method comprises modifying a target
polynucleotide using a CRISPR complex that binds to the target
polynucleotide and effect cleavage of said target polynucleotide.
Typically, the CRISPR complex of the invention, when introduced
into a cell, creates a break (e.g., a single or a double strand
break) in the genome sequence. For example, the method can be used
to cleave a disease gene in a cell. The break created by the CRISPR
complex can be repaired by a repair processes such as the error
prone non-homologous end joining (NHEJ) pathway or the high
fidelity homology-directed repair (HDR). During these repair
process, an exogenous polynucleotide template can be introduced
into the genome sequence. In some methods, the HDR process is used
modify genome sequence. For example, an exogenous polynucleotide
template comprising a sequence to be integrated flanked by an
upstream sequence and a downstream sequence is introduced into a
cell. The upstream and downstream sequences share sequence
similarity with either side of the site of integration in the
chromosome. Where desired, a donor polynucleotide can be DNA, e.g.,
a DNA plasmid, a bacterial artificial chromosome (BAC), a yeast
artificial chromosome (YAC), a viral vector, a linear piece of DNA,
a PCR fragment, a naked nucleic acid, or a nucleic acid complexed
with a delivery vehicle such as a liposome or poloxamer. The
exogenous polynucleotide template comprises a sequence to be
integrated (e.g., a mutated gene). The sequence for integration may
be a sequence endogenous or exogenous to the cell. Examples of a
sequence to be integrated include polynucleotides encoding a
protein or a non-coding RNA (e.g., a microRNA). Thus, the sequence
for integration may be operably linked to an appropriate control
sequence or sequences. Alternatively, the sequence to be integrated
may provide a regulatory function. The upstream and downstream
sequences in the exogenous polynucleotide template are selected to
promote recombination between the chromosomal sequence of interest
and the donor polynucleotide. The upstream sequence is a nucleic
acid sequence that shares sequence similarity with the genome
sequence upstream of the targeted site for integration. Similarly,
the downstream sequence is a nucleic acid sequence that shares
sequence similarity with the chromosomal sequence downstream of the
targeted site of integration. The upstream and downstream sequences
in the exogenous polynucleotide template can have 75%, 80%, 85%,
90%, 95%, or 100% sequence identity with the targeted genome
sequence. Preferably, the upstream and downstream sequences in the
exogenous polynucleotide template have about 95%, 96%, 97%, 98%,
99%, or 100% sequence identity with the targeted genome sequence.
In some methods, the upstream and downstream sequences in the
exogenous polynucleotide template have about 99% or 100% sequence
identity with the targeted genome sequence. An upstream or
downstream sequence may comprise from about 20 bp to about 2500 bp,
for example, about 50, 100, 200, 300, 400, 500, 600, 700, 800, 900,
1000, 1100, 1200, 1300, 1400, 1500, 1600, 1700, 1800, 1900, 2000,
2100, 2200, 2300, 2400, or 2500 bp. In some methods, the exemplary
upstream or downstream sequence have about 200 bp to about 2000 bp,
about 600 bp to about 1000 bp, or more particularly about 700 bp to
about 1000 bp. In some methods, the exogenous polynucleotide
template may further comprise a marker. Such a marker may make it
easy to screen for targeted integrations. Examples of suitable
markers include restriction sites, fluorescent proteins, or
selectable markers. The exogenous polynucleotide template of the
invention can be constructed using recombinant techniques (see, for
example, Sambrook et al., 2001 and Ausubel et al., 1996). In a
method for modifying a target polynucleotide by integrating an
exogenous polynucleotide template, a double stranded break is
introduced into the genome sequence by the CRISPR complex, the
break is repaired via homologous recombination an exogenous
polynucleotide template such that the template is integrated into
the genome. The presence of a double-stranded break facilitates
integration of the template. In other embodiments, this invention
provides a method of modifying expression of a polynucleotide in a
eukaryotic cell. The method comprises increasing or decreasing
expression of a target polynucleotide by using a CRISPR complex
that binds to the polynucleotide. In some methods, a target
polynucleotide can be inactivated to effect the modification of the
expression in a cell. For example, upon the binding of a CRISPR
complex to a target sequence in a cell, the target polynucleotide
is inactivated such that the sequence is not transcribed, the coded
protein is not produced, or the sequence does not function as the
wild-type sequence does. For example, a protein or microRNA coding
sequence may be inactivated such that the protein or microRNA or
pre-microRNA transcript is not produced. In some methods, a control
sequence can be inactivated such that it no longer functions as a
control sequence. As used herein, "control sequence" refers to any
nucleic acid sequence that effects the transcription, translation,
or accessibility of a nucleic acid sequence. Examples of a control
sequence include, a promoter, a transcription terminator, and an
enhancer are control sequences. The target polynucleotide of a
CRISPR complex can be any polynucleotide endogenous or exogenous to
the eukaryotic cell. For example, the target polynucleotide can be
a polynucleotide residing in the nucleus of the eukaryotic cell.
The target polynucleotide can be a sequence coding a gene product
(e.g., a protein) or a non-coding sequence (e.g., a regulatory
polynucleotide or a junk DNA). Examples of target polynucleotides
include a sequence associated with a signaling biochemical pathway,
e.g., a signaling biochemical pathway-associated gene or
polynucleotide. Examples of target polynucleotides include a
disease associated gene or polynucleotide. A "disease-associated"
gene or polynucleotide refers to any gene or polynucleotide which
is yielding transcription or translation products at an abnormal
level or in an abnormal form in cells derived from a
disease-affected tissues compared with tissues or cells of a non
disease control. It may be a gene that becomes expressed at an
abnormally high level; it may be a gene that becomes expressed at
an abnormally low level, where the altered expression correlates
with the occurrence and/or progression of the disease. A
disease-associated gene also refers to a gene possessing
mutation(s) or genetic variation that is directly responsible or is
in linkage disequilibrium with a gene(s) that is responsible for
the etiology of a disease. The transcribed or translated products
may be known or unknown, and may be at a normal or abnormal level.
The target polynucleotide of a CRISPR complex can be any
polynucleotide endogenous or exogenous to the eukaryotic cell. For
example, the target polynucleotide can be a polynucleotide residing
in the nucleus of the eukaryotic cell. The target polynucleotide
can be a sequence coding a gene product (e.g., a protein) or a
non-coding sequence (e.g., a regulatory polynucleotide or a junk
DNA).
[0134] The target polynucleotide of a CRISPR complex can be any
polynucleotide endogenous or exogenous to the eukaryotic cell. For
example, the target polynucleotide can be a polynucleotide residing
in the nucleus of the eukaryotic cell. The target polynucleotide
can be a sequence coding a gene product (e.g., a protein) or a
non-coding sequence (e.g., a regulatory polynucleotide or a junk
DNA). The target can be a control element or a regulatory element
or a promoter or an enhancer or a silencer. The promoter may, in
some embodiments, be in the region of +200 bp or even +1000 bp from
the TTS. In some embodiments, the regulatory region may be an
enhancer. The enhancer is typically more than +1000 bp from the
TTS. More in particular, expression of eukaryotic protein-coding
genes generally is regulated through multiple cis-acting
transcription-control regions. Some control elements are located
close to the start site (promoter-proximal elements), whereas
others lie more distant (enhancers and silencers) Promoters
determine the site of transcription initiation and direct binding
of RNA polymerase II. Three types of promoter sequences have been
identified in eukaryotic DNA. The TATA box, the most common, is
prevalent in rapidly transcribed genes. Initiator promoters
infrequently are found in some genes, and CpG islands are
characteristic of transcribed genes. Promoter-proximal elements
occur within .apprxeq.200 base pairs of the start site. Several
such elements, containing up to .apprxeq.20 base pairs, may help
regulate a particular gene. Enhancers, which are usually
.apprxeq.100-200 base pairs in length, contain multiple 8- to 20-bp
control elements. They may be located from 200 base pairs to tens
of kilobases upstream or downstream from a promoter, within an
intron, or downstream from the final exon of a gene.
Promoter-proximal elements and enhancers may be cell-type specific,
functioning only in specific differentiated cell types. However,
any of these regions can be the target sequence and are encompassed
by the concept that the target can be a control element or a
regulatory element or a promoter or an enhancer or a silencer.
[0135] Without wishing to be bound by theory, it is believed that
the target sequence should be associated with a PAM (protospacer
adjacent motif); that is, a short sequence recognized by the CRISPR
complex. The precise sequence and length requirements for the PAM
differ depending on the CRISPR enzyme used, but PAMs are typically
2-5 base pair sequences adjacent the protospacer (that is, the
target sequence) Examples of PAM sequences are given in the
examples section below, and the skilled person will be able to
identify further PAM sequences for use with a given CRISPR enzyme.
In some embodiments, the method comprises allowing a CRISPR complex
to bind to the target polynucleotide to effect cleavage of said
target polynucleotide thereby modifying the target polynucleotide,
wherein the CRISPR complex comprises a CRISPR enzyme complexed with
a guide sequence hybridized to a target sequence within said target
polynucleotide, wherein said guide sequence is linked to a tracr
mate sequence which in turn hybridizes to a tracr sequence. In one
aspect, the invention provides a method of modifying expression of
a polynucleotide in a eukaryotic cell. In some embodiments, the
method comprises allowing a CRISPR complex to bind to the
polynucleotide such that said binding results in increased or
decreased expression of said polynucleotide; wherein the CRISPR
complex comprises a CRISPR enzyme complexed with a guide sequence
hybridized to a target sequence within said polynucleotide, wherein
said guide sequence is linked to a tracr mate sequence which in
turn hybridizes to a tracr sequence. Similar considerations and
conditions apply as above for methods of modifying a target
polynucleotide. In fact, these sampling, culturing and
re-introduction options apply across the aspects of the present
invention. In one aspect, the invention provides for methods of
modifying a target polynucleotide in a eukaryotic cell, which may
be in vivo, ex vivo or in vitro. In some embodiments, the method
comprises sampling a cell or population of cells from a human or
non-human animal, and modifying the cell or cells. Culturing may
occur at any stage ex vivo. The cell or cells may even be
re-introduced into the non-human animal or plant. For re-introduced
cells it is particularly preferred that the cells are stem
cells.
[0136] Indeed, in any aspect of the invention, the CRISPR complex
may comprise a CRISPR enzyme complexed with a guide sequence
hybridized to a target sequence, wherein said guide sequence may be
linked to a tracr mate sequence which in turn may hybridize to a
tracr sequence.
[0137] The invention relates to the engineering and optimization of
systems, methods and compositions used for the control of gene
expression involving sequence targeting, such as genome
perturbation or gene-editing, that relate to the CRISPR-Cas system
and components thereof. The Cas enzyme is Cas9. An advantage of the
present methods is that the CRISPR system minimizes or avoids
off-target binding and its resulting side effects. This is achieved
using systems arranged to have a high degree of sequence
specificity for the target DNA.
[0138] In relation to a CRISPR-Cas complex or system preferably,
the tracr sequence has one or more hairpins and is 30 or more
nucleotides in length, 40 or more nucleotides in length, or 50 or
more nucleotides in length; the guide sequence is between 10 to 30
nucleotides in length, the CRISPR/Cas enzyme is a Type II Cas9
enzyme.
[0139] In one aspect, the invention provides a non-naturally
occurring or engineered CRISPR enzyme associated with at least one
destabilization domain (DD); and, for shorthand purposes, such a
non-naturally occurring or engineered CRISPR enzyme associated with
at least one destabilization domain (DD) is herein termed a
"DD-CRISPR enzyme". In one aspect, the invention provides an
engineered, non-naturally occurring DD-CRISPR-Cas system comprising
a DD-CRISPR enzyme, wherein the CRISPR enzyme is a Cas protein
(herein termed a "DD-Cas protein"), which is a type II DD-CRISPR
Cas9 protein associated with at least one destabilization domain
(herein termed a "DD-Cas9 protein" (or simply "DD-Cas9") and guide
RNA that targets a nucleic acid molecule such as a DNA molecule,
whereby the guide RNA targets the nucleic acid molecule, e.g., DNA
molecule. The nucleic acid molecule, e.g., DNA molecule can encode
a gene product. In some embodiments the DD-Cas protein may cleave
the DNA molecule encoding the gene product. In some embodiments
expression of the gene product is altered. The Cas protein and the
guide RNA do not naturally occur together. The invention
comprehends the guide RNA comprising a guide sequence fused to a
tracr sequence. The invention further comprehends coding for the
Cas protein being codon optimized for expression in a eukaryotic
cell. In a preferred embodiment the eukaryotic cell is a mammalian
cell and in a more preferred embodiment the mammalian cell is a
human cell. Expression of the gene product may be decreased. The
CRISPR enzyme may form part of a CRISPR-Cas system, which further
comprises a guide RNA (sgRNA) comprising a guide sequence capable
of hybridizing to a target sequence in a genomic locus of interest
in a cell. In some embodiments, the functional CRISPR-Cas system
binds to the target sequence. In some embodiments, the functional
CRISPR-Cas system may edit the target sequence, e.g., the target
sequence may comprise a genomic locus, and in some embodiments
there may be an alteration of gene expression. In some embodiments,
the functional CRISPR-Cas system may comprise further functional
domains. In some embodiments, the invention provides a method for
altering or modifying expression of a gene product. The method may
comprise introducing into a cell containing a target nucleic acid,
e.g., DNA molecule, or containing and expressing a target nucleic
acid, e.g., DNA molecule; for instance, the target nucleic acid may
encode a gene product or provide for expression of a gene product
(e.g., a regulatory sequence).
[0140] The DD-CRISPR enzyme is a DD-Cas9. In some embodiments, the
DD-CRISPR enzyme is an Sp DD-Cas9. In some embodiments, the CRISPR
enzyme is an Sa DD-Cas9. In some embodiments, the CRISPR enzyme is
an St or Fn DD-Cas9, although other orthologs are envisaged. Sp and
Sa DD-Cas9s are particularly preferred, in some embodiments. In
some embodiments, the DD-CRISPR enzyme cleave both strands of DNA
to produce a double strand break (DSB). In some embodiments, the
DD-CRISPR enzyme is a nickase. In some embodiments, the DD-CRISPR
enzyme is a dual nickase. In some embodiments, the DD-CRISPR enzyme
is a deadCas9, e.g., a Cas9 having substantially no nuclease
activity, e.g., no more than 5% nuclease activity as compared with
a wild-type Cas9 or Cas9 not having had mutations to it.
[0141] In some general embodiments, the DD-CRISPR enzyme is
associated with one or more functional domains. In some more
specific embodiments, the DD-CRISPR enzyme is a deadCas9 and/or is
associated with one or more functional domains.
[0142] In some embodiments, the DD-CRISPR enzyme comprises a Rec2
or HD2 truncation. In some embodiments, the CRISPR enzyme is
associated with the DD by way of a fusion protein. In some
embodiments, the CRISPR enzyme is fused to the DD. In other words,
the DD may be associated with the CRISPR enzyme by fusion with said
CRISPR enzyme. In some embodiments, the enzyme may be considered to
be a modified CRISPR enzyme, wherein the CRISPR enzyme is fused to
at least one destabilization domain (DD). In some embodiments, the
DD may be associated to the CRISPR enzyme via a connector protein,
for example using a system such as a marker system such as the
streptavidin-biotin system. As such, provided is a fusion of a
CRISPR enzyme with a connector protein specific for a high affinity
ligand for that connector, whereas the DD is bound to said high
affinity ligand. For example, streptavidin may be the connector
fused to the CRISPR enzyme, while biotin may be bound to the DD.
Upon co-localization, the streptavidin will bind to the biotin,
thus connecting the CRISPR enzyme to the DD. For simplicity, a
fusion of the CRISPR enzyme and the DD is preferred in some
embodiments. In some embodiments, the fusion may be to the
N-terminal end of the CRISPR enzyme. In some embodiments, at least
one DD is fused to the N-terminus of the CRISPR enzyme. In some
embodiments, the fusion may be to the C-terminal end of the CRISPR
enzyme. In some embodiments, at least one DD is fused to the
C-terminus of the CRISPR enzyme. In some embodiments, one DD may be
fused to the N-terminal end of the CRISPR enzyme with another DD
fused to the C-terminal of the CRISPR enzyme. In some embodiments,
the CRISPR enzyme is associated with at least two DDs and wherein a
first DD is fused to the N-terminus of the CRISPR enzyme and a
second DD is fused to the C-terminus of the CRISPR enzyme, the
first and second DDs being the same or different. In some
embodiments, the fusion may be to the N-terminal end of the DD. In
some embodiments, the fusion may be to the C-terminal end of the
DD. In some embodiments, the fusion may between the C-terminal end
of the CRISPR enzyme and the N-terminal end of the DD. In some
embodiments, the fusion may between the C-terminal end of the DD
and N-terminal end of the CRISPR enzyme. Less background was
observed with a DD comprising at least one N-terminal fusion than a
DD comprising at least one C terminal fusion. Combining N- and
C-terminal fusions had the least background but lowest overall
activity. Advantageously a DD is provided through at least one
N-terminal fusion or at least one N terminal fusion plus at least
one C-terminal fusion. And of course, a DD can be provided by at
least one C-terminal fusion.
[0143] In some embodiments, the DD is ER50. A corresponding
stabilizing ligand for this DD is, in some embodiments, 4HT. As
such, in some embodiments, one of the at least one DDs is ER50 and
a stabilizing ligand therefor is 4HT or CMP8. In some embodiments,
the DD is DHFR50. A corresponding stabilizing ligand for this DD
is, in some embodiments, TMP. As such, in some embodiments, one of
the at least one DDs is DHFR50 and a stabilizing ligand therefor is
TMP. In some embodiments, the DD is ER50. A corresponding
stabilizing ligand for this DD is, in some embodiments, CMP8. CMP8
may therefore be an alternative stabilizing ligand to 4HT in the
ER50 system. While it may be possible that CMP8 and 4HT can/should
be used in a competitive matter, some cell types may be more
susceptible to one or the other of these two ligands, and from this
disclosure and the knowledge in the art the skilled person can use
CMP8 and/or 4HT.
[0144] In some embodiments, one or two DDs may be fused to the
N-terminal end of the CRISPR enzyme with one or two DDs fused to
the C-terminal of the CRISPR enzyme. In some embodiments, the at
least two DDs are associated with the CRISPR enzyme and the DDs are
the same DD, i.e. the DDs are homologous. Thus, both (or two or
more) of the DDs could be ER50 DDs. This is preferred in some
embodiments. Alternatively, both (or two or more) of the DDs could
be DHFR50 DDs. This is also preferred in some embodiments. In some
embodiments, the at least two DDs are associated with the CRISPR
enzyme and the DDs are different DDs, i.e. the DDs are
heterologous. Thus, one of the DDS could be ER50 while one or more
of the or any other DDs could be DHFR50. Having two or more DDs
which are heterologous may be advantageous as it would provide a
greater level of degradation control. A tandem fusion of more than
one DD at the N or C-term may enhance degradation; and such a
tandem fusion can be, for example ER50-ER50-Cas9 or DHFR-DHFR-Ca9
It is envisaged that high levels of degradation would occur in the
absence of either stabilizing ligand, intermediate levels of
degradation would occur in the absence of one stabilizing ligand
and the presence of the other (or another) stabilizing ligand,
while low levels of degradation would occur in the presence of both
(or two of more) of the stabilizing ligands. Control may also be
imparted by having an N-terminal ER50 DD and a C-terminal DHFR50
DD.
[0145] In some embodiments, the fusion of the CRISPR enzyme with
the DD comprises a linker between the DD and the CRISPR enzyme. In
some embodiments, the linker is a GlySer linker. In some
embodiments, the DD-CRISPR enzyme further comprises at least one
Nuclear Export Signal (NES). In some embodiments, the DD-CRISPR
enzyme comprises two or more NESs. In some embodiments, the
DD-CRISPR enzyme comprises at least one Nuclear Localization Signal
(NLS). This may be in addition to an NES. In some embodiments, the
CRISPR enzyme comprises or consists essentially of or consists of a
localization (nuclear import or export) signal as, or as part of,
the linker between the CRISPR enzyme and the DD. HA or Flag tags
are also within the ambit of the invention as linkers. Applicants
use NLS and/or NES as linker and use Glycine Serine linkers as
short as GS up to (GGGGS)3 (SEQ ID NO: 27).
[0146] In an aspect, the present invention provides a
polynucleotide encoding the CRISPR enzyme and associated DD. In
some embodiments, the encoded CRISPR enzyme and associated DD are
operably linked to a first regulatory element. In some embodiments,
a DD is also encoded and is operably linked to a second regulatory
element. Advantageously, the DD here is to "mop up" the stabilizing
ligand and so it is advantageously the same DD (i.e. the same type
of Domain) as that associated with the enzyme, e.g., as herein
discussed (with it understood that the term "mop up" is meant in
the sense of performing so as to contribute or conclude activity.
In some embodiments, the first regulatory element is a promoter and
may optionally include an enhancer. In some embodiments, the second
regulatory element is a promoter and may optionally include an
enhancer. In some embodiments, the first regulatory element is an
early promoter. In some embodiments, the second regulatory element
is a late promoter. In some embodiments, the second regulatory
element is or comprises or consists essentially of an inducible
control element, optionally the tet system, or a repressible
control element, optionally the tetr system. An inducible promoter
may be favorable e.g. rTTA to induce tet in the presence of
doxycycline.
[0147] In an aspect, the present invention provides a means for
delivering the DD-CRISPR-Cas complex of the invention or
polynucleotides discussed herein, e.g., particle(s) delivering
component(s) of the complex, vector(s) comprising the
polynucleotide(s) discussed herein (e.g., encoding the CRISPR
enzyme, the DD; providing RNA of the CRISPR-Cas complex). In some
embodiments, the vector may be a plasmid or a viral vector such as
AAV, or lentivirus. Transient transfection with plasmids, e.g.,
into HEK cells may be advantageous, especially given the size
limitations of AAV and that while SpCas9 fits into AAV, one may
reach an upper limit with additional coding as to the association
with the DD(s).
[0148] Also provided is a model that constitutively expresses the
CRISPR enzyme and associated DD. The organism may be a transgenic
and may have been transfected the present vectors or may be the
offspring of an organism so transfected. In a further aspect, the
present invention provides compositions comprising the CRISPR
enzyme and associated DD or the polynucleotides or vectors
described herein. Also provides are CRISPR-Cas systems comprising
guide RNAs.
[0149] Also provided is a method of treating a subject, e.g., a
subject in need thereof, comprising inducing gene editing by
transforming the subject with the polynucleotide encoding the
system or any of the present vectors and administering stabilizing
ligand to the subject. A suitable repair template may also be
provided, for example delivered by a vector comprising said repair
template. Also provided is a method of treating a subject, e.g., a
subject in need thereof, comprising inducing transcriptional
activation or repression by transforming the subject with the
polynucleotide encoding the present system or any of the present
vectors, wherein said polynucleotide or vector encodes or comprises
the catalytically inactive CRISPR enzyme and one or more associated
functional domains; the method further comprising administering a
stabilizing ligand to the subject. These methods may also include
delivering and/or expressing excess DD to the subject. Where any
treatment is occurring ex vivo, for example in a cell culture, then
it will be appreciated that the term `subject` may be replaced by
the phrase "cell or cell culture."
[0150] Compositions comprising the present system for use in said
method of treatment are also provided. A separate composition may
comprise the stabilizing ligand. A kit of parts may be provided
including such compositions. Use of the present system in the
manufacture of a medicament for such methods of treatment are also
provided. Use of the present system in screening is also provided
by the present invention, e.g., gain of function screens. Cells
which are artificially forced to overexpress a gene are be able to
down regulate the gene over time (re-establishing equilibrium) e.g.
by negative feedback loops. By the time the screen starts the
unregulated gene might be reduced again. Using an inducible Cas9
activator allows one to induce transcription right before the
screen and therefore minimizes the chance of false negative hits.
Accordingly, by use of the instant invention in screening, e.g.,
gain of function screens, the chance of false negative results may
be minimized.
[0151] In one aspect, the invention provides an engineered,
non-naturally occurring CRISPR-Cas system comprising a DD-Cas9
protein and a guide RNA that targets a DNA molecule encoding a gene
product in a cell, whereby the guide RNA targets the DNA molecule
encoding the gene product and the Cas9 protein cleaves the DNA
molecule encoding the gene product, whereby expression of the gene
product is altered; and, wherein the Cas9 protein and the guide RNA
do not naturally occur together. The invention comprehends the
guide RNA comprising a guide sequence fused to a tracr sequence.
The Cas protein is a type II CRISPR-Cas protein and is a Cas9
protein. The invention further comprehends coding for the Cas9
protein being codon optimized for expression in a eukaryotic cell.
In a preferred embodiment the eukaryotic cell is a mammalian cell
and in a more preferred embodiment the mammalian cell is a human
cell. In a further embodiment of the invention, the expression of
the gene product is decreased.
[0152] Where functional domains and the like are "associated" with
one or other part of the enzyme, these are typically fusions. The
term "associated with" is used here in respect of how one molecule
`associates` with respect to another, for example between parts of
the CRISPR enzyme an a functional domain. The two may be considered
to be tethered to each other. In the case of such protein-protein
interactions, this association may be viewed in terms of
recognition in the way an antibody recognizes an epitope.
Alternatively, one protein may be associated with another protein
via a fusion of the two, for instance one subunit being fused to
another subunit. Fusion typically occurs by addition of the amino
acid sequence of one to that of the other, for instance via
splicing together of the nucleotide sequences that encode each
protein or subunit. Alternatively, this may essentially be viewed
as binding between two molecules or direct linkage, such as a
fusion protein. In any event, the fusion protein may include a
linker between the two subunits of interest (e.g. between the
enzyme and the functional domain or between the adaptor protein and
the functional domain). Thus, in some embodiments, the part of the
CRISPR enzyme is associated with a functional domain by binding
thereto. In other embodiments, the CRISPR enzyme is associated with
a functional domain because the two are fused together, optionally
via an intermediate linker. Examples of linkers include the GlySer
linkers discussed herein. While a non covalent bound DD may be able
to initiate degradation of the associated Cas9, proteasome
degradation involves unwinding of the protein chain; and, a fusion
is preferred as it can provide that the DD stays connected to Cas9
upon degradation. However the CRISPR enzyme and DD are brought
together, in the presence of a stabilizing ligand specific for the
DD, a stabilization complex is formed. This complex comprises the
stabilizing ligand bound to the DD. The complex also comprises the
DD associated with the CRISPR enzyme. In the absence of said
stabilizing ligand, degradation of the DD and its associated CRISPR
enzyme is promoted.
[0153] Destabilizing domains have general utility to confer
instability to a wide range of proteins; see, e.g., Miyazaki, J Am
Chem Soc. Mar. 7, 2012; 134(9): 3942-3945, and Chung H Nature
Chemical Biology Vol. 11 Sep. 2015 pgs 713-720, incorporated herein
by reference. CMP8 or 4-hydroxytamoxifen can be destabilizing
domains. More generally, a temperature-sensitive mutant of
mammalian DHFR (DHFRts), a destabilizing residue by the N-end rule,
was found to be stable at a permissive temperature but unstable at
37.degree. C. The addition of methotrexate, a high-affinity ligand
for mammalian DHFR, to cells expressing DHFRts inhibited
degradation of the protein partially. This was an important
demonstration that a small molecule ligand can stabilize a protein
otherwise targeted for degradation in cells. A rapamycin derivative
was used to stabilize an unstable mutant of the FRB domain of mTOR
(FRB*) and restore the function of the fused kinase,
GSK-3.beta..6,7 This system demonstrated that ligand-dependent
stability represented an attractive strategy to regulate the
function of a specific protein in a complex biological environment.
A system to control protein activity can involve the DD becoming
functional when the ubiquitin complementation occurs by rapamycin
induced dimerization of FK506-binding protein and FKBP12. Mutants
of human FKBP12 or ecDHFR protein can be engineered to be
metabolically unstable in the absence of their high-affinity
ligands, Shield-1 or trimethoprim (TMP), respectively. These
mutants are some of the possible destabilizing domains (DDs) useful
in the practice of the invention and instability of a DD as a
fusion with a CRISPR enzyme confers to the CRISPR protein
degradation of the entire fusion protein by the proteasome.
Shield-1 and TMP bind to and stabilize the DD in a dose-dependent
manner. The estrogen receptor ligand binding domain (ERLBD,
residues 305-549 of ERS1) can also be engineered as a destabilizing
domain. Since the estrogen receptor signaling pathway is involved
in a variety of diseases such as breast cancer, the pathway has
been widely studied and numerous agonist and antagonists of
estrogen receptor have been developed. Thus, compatible pairs of
ERLBD and drugs are known. There are ligands that bind to mutant
but not wild-type forms of the ERLBD. By using one of these mutant
domains encoding three mutations (L384M, M421G, G521R)12, it is
possible to regulate the stability of an ERLBD-derived DD using a
ligand that does not perturb endogenous estrogen-sensitive
networks. An additional mutation (Y537S) can be introduced to
further destabilize the ERLBD and to configure it as a potential DD
candidate. This tetra-mutant is an advantageous DD development. The
mutant ERLBD can be fused to a CRISPR enzyme and its stability can
be regulated or perturbed using a ligand, whereby the CRISPR enzyme
has a DD. Another DD can be a 12-kDa (107-amino-acid) tag based on
a mutated FKBP protein, stabilized by Shield1 ligand; see, e.g.,
Nature Methods 5, (2008). For instance a DD can be a modified FK506
binding protein 12 (FKBP12) that binds to and is reversibly
stabilized by a synthetic, biologically inert small molecule,
Shield-1; see, e.g., Banaszynski L A, Chen L C, Maynard-Smith L A,
Ooi A G, Wandless T J. A rapid, reversible, and tunable method to
regulate protein function in living cells using synthetic small
molecules. Cell. 2006; 126:995-1004; Banaszynski L A, Sellmyer M A,
Contag C H, Wandless T J, Thorne S H. Chemical control of protein
stability and function in living mice. Nat Med. 2008; 14:1123-1127;
Maynard-Smith L A, Chen L C, Banaszynski L A, Ooi A G, Wandless T
J. A directed approach for engineering conditional protein
stability using biologically silent small molecules. The Journal of
biological chemistry. 2007; 282:24866-24872; and Rodriguez, Chem
Biol. Mar. 23, 2012; 19(3): 391-398--all of which are incorporated
herein by reference and may be employed in the practice of the
invention in selected a DD to associate with a CRISPR enzyme in the
practice of this invention. As can be seen, the knowledge in the
art includes a number of DDs, and the DD can be associated with,
e.g., fused to, advantageously with a linker, to a CRISPR enzyme,
whereby the DD can be stabilized in the presence of a ligand and
when there is the absence thereof the DD can become destabilized,
whereby the CRISPR enzyme is entirely destabilized, or the DD can
be stabilized in the absence of a ligand and when the ligand is
present the DD can become destabilized; the DD allows the CRISPR
enzyme and hence the CRISPR-Cas complex or system to be regulated
or controlled--turned on or off so to speak, to thereby provide
means for regulation or control of the system, e.g., in an in vivo
or in vitro environment. For instance, when a protein of interest
is expressed as a fusion with the DD tag, it is destabilized and
rapidly degraded in the cell, e.g., by proteasomes. Thus, absence
of stabilizing ligand leads to a D associated Cas9 being degraded.
When a new DD is fused to a protein of interest, its instability is
conferred to the protein of interest, resulting in the rapid
degradation of the entire fusion protein. Peak activity for Cas9 is
sometimes beneficial to reduce off-target effects. Thus, short
bursts of high activity are preferred. The present invention is
able to provide such peaks. In some senses the system is inducible.
In some other senses, the system repressed in the absence of
stabilizing ligand and de-repressed in the presence of stabilizing
ligand. Without wishing to be bound by any theory and without
making any promises, other benefits of the invention may include
that it is: [0154] Dosable (in contrast to a system that turns on
or off, e.g., can allow for variable CRISPR-Cas system or complex
activity). [0155] Orthogonal, e.g., a ligand only affects its
cognate DD so two or more systems can operate independently, and/or
the CRISPR enzymes can be from one or more orthologs. [0156]
Transportable, e.g., may work in different cell types or cell
lines. [0157] Rapid. [0158] Temporal Control. [0159] Able to reduce
background or off target Cas9 or Cas9 toxicity or excess build up
of Cas9 by allowing the Cas9 to be degraded. [0160] Cheap--the
stabilizing ligands are widely available and typically not
expensive.
[0161] While the DD can be at N and/or C terminal(s) of the Cas9 or
CRISPR enzyme, including a DD at one or more sides of a split e.g.
Cas9(N)-linker-DD-linker-Cas9(C) is also a way to introduce a DD.
In some embodiments, if using only one terminal association of DD
to the CRISPR enzyme is to be used, then it is preferred to use
ER50 as the DD. In some embodiments, if using both N- and
C-terminals, then use of either ER50 and/or DHFR50 is preferred.
Particularly good results were seen with the N-terminal fusion,
which is surprising. Having both N and C terminal fusion may be
synergistic. The size of Destabilization Domain varies but is
typically approx.--approx. 100-300 amino acids in size. The DD is
preferably an engineered destabilizing protein domain. DDs and
methods for making DDs, e.g., from a high affinity ligand and its
ligand binding domain. The invention may be considered to be
"orthogonal" as only the specific ligand will stabilize its
respective (cognate) DD, it will have no effect on the stability of
non-cognate DDs. A commercially available DD system is the
CloneTech, ProteoTuner.TM. system; the stabilizing ligand is
Shield1, which is also preferred for use in the present
invention.
[0162] In some embodiments, the stabilizing ligand is a `small
molecule`. In some embodiments, the stabilizing ligand is
cell-permeable. It has a high affinity for its corresponding DD.
Suitable DD--stabilizing ligand pairs are known in the art. In
general, the stabilizing ligand may be removed by: [0163] Natural
processing (e.g., proteasome degradation), e.g., in vivo; [0164]
Mopping up, e.g. ex vivo/cell culture, by: [0165] Provision of a
preferred binding partner; or
[0166] Provision of XS Substrate (DD without Cas9),
[0167] Advantageously, the DD may "mop up" the stabilizing ligand
and so it is the same DD (i.e. the same type of DD) as that
associated with the enzyme. By mopping up the stabilizing ligand
with excess DD that is not associated with the CRISPR enzyme,
greater degradation of the CRISPR enzyme will be seen. It is
envisaged, without being bound by theory, that as additional or
excess un-associated DD is added that the equilibrium will shift
away from the stabilizing ligand complexing or binding to the DD
associated with the CRISPR enzyme and instead move towards more of
the stabilizing ligand complexing or binding to the free DD (i.e.
that not associated with the CRISPR enzyme). Thus, provision of
excess or additional unassociated (or free) DD is preferred when it
is desired to reduce CRISPR enzyme activity through increased
degradation of the CRISPR enzyme. An excess of free DD will bind
residual ligand and also takes away bound ligand from DD-Cas9
fusion. Therefore it accelerates DD-Cas9 degradation and enhances
temporal control of Cas9 activity.
[0168] The present invention also contemplates use of the
CRISPR-Cas9/ and Destabilization Domain (DD)-Cas9 system described
herein to provide RNA-guided gene drives, for example in systems
analogous to gene drives described in PCT Patent Publication WO
2015/105928. Systems of this kind may for example provide methods
for altering eukaryotic germline cells, by introducing into the
germline cell a nucleic acid sequence encoding an RNA-guided DNA
nuclease and one or more guide RNAs. The guide RNAs may be designed
to be complementary to one or more target locations on genomic DNA
of the germline cell. The nucleic acid sequence encoding the RNA
guided DNA nuclease and the nucleic acid sequence encoding the
guide RNAs may be provided on constructs between flanking
sequences, with promoters arranged such that the germline cell may
express the RNA guided DNA nuclease and the guide RNAs, together
with any desired cargo-encoding sequences that are also situated
between the flanking sequences. The flanking sequences will
typically include a sequence which is identical to a corresponding
sequence on a selected target chromosome, so that the flanking
sequences work with the components encoded by the construct to
facilitate insertion of the foreign nucleic acid construct
sequences into genomic DNA at a target cut site by mechanisms such
as homologous recombination, to render the germline cell homozygous
for the foreign nucleic acid sequence. In this way, gene-drive
systems are capable of introgressing desired cargo genes throughout
a breeding population (Gantz et al., 2015, Highly efficient
Cas9-mediated gene drive for population modification of the malaria
vector mosquito Anopheles stephensi, PNAS 2015, published ahead of
print Nov. 23, 2015, doi:10.1073/pnas.1521077112; Esvelt et al.,
2014, Concerning RNA-guided gene drives for the alteration of wild
populations eLife 2014; 3:e03401). In select embodiments, target
sequences may be selected which have few potential off-target sites
in a genome. Targeting multiple sites within a target locus, using
multiple guide RNAs, may increase the cutting frequency and hinder
the evolution of drive resistant alleles. Truncated guide RNAs may
reduce off-target cutting. Paired nickases may be used instead of a
single nuclease, to further increase specificity. Gene drive
constructs may include cargo sequences encoding transcriptional
regulators, for example to activate homologous recombination genes
and/or repress non-homologous end-joining. Target sites may be
chosen within an essential gene, so that non-homologous end-joining
events may cause lethality rather than creating a drive-resistant
allele. The gene drive constructs can be engineered to function in
a range of hosts at a range of temperatures (Cho et al. 2013, Rapid
and Tunable Control of Protein Stability in Caenorhabditis elegans
Using a Small Molecule, PLoS ONE 8(8): e72393.
doi:10.1371/journal.pone.0072393).
[0169] In order to enforce the gene drive, then the stabilizing
ligand will need to be present in the environment (such as the
diet) of the host. In the absence of a stabilizing ligand, the Cas9
will be degraded and no or reduced gene drive will be seen.
[0170] Attachment or association can be via a linker, e.g., a
flexible glycine-serine (GlyGlyGlySer) (SEQ ID NO: 28) or
(GGGS).sub.3 (SEQ ID NO: 29) or a rigid alpha-helical linker such
as (Ala(GluAlaAlaAlaLys)Ala) (SEQ ID NO: 30). Linkers such as
(GGGGS).sub.3 (SEQ ID NO: 27) are preferably used herein to
separate protein or peptide domains. (GGGGS).sub.3 (SEQ ID NO: 27)
is preferable because it is a relatively long linker (15 amino
acids). The glycine residues are the most flexible and the serine
residues enhance the chance that the linker is on the outside of
the protein. (GGGGS).sub.6 (SEQ ID NO: 31) (GGGGS).sub.9 (SEQ ID
NO: 32) or (GGGGS).sub.12 (SEQ ID NO: 33) may preferably be used as
alternatives. Other preferred alternatives are (GGGGS).sub.1 (SEQ
ID NO: 34), (GGGGS).sub.2 (SEQ ID NO: 35), (GGGGS).sub.4 (SEQ ID
NO: 36), (GGGGS).sub.5 (SEQ ID NO: 37), (GGGGS).sub.7 (SEQ ID NO:
38), (GGGGS).sub.8 (SEQ ID NO: 39), (GGGGS).sub.10 (SEQ ID NO: 40),
or (GGGGS).sub.11 (SEQ ID NO: 41). Alternative linkers are
available, but highly flexible linkers are thought to work best to
allow for maximum opportunity for the 2 parts of the Cas9 to come
together and thus reconstitute Cas9 activity. One alternative is
that the NLS of nucleoplasmin can be used as a linker. For example,
a linker can also be used between the Cas9 and any functional
domain. Again, a (GGGGS).sub.3 linker (SEQ ID NO: 27) may be used
here (or the 6, 9, or 12 repeat versions therefore) or the NLS of
nucleoplasmin can be used as a linker between Cas9 and the
functional domain.
[0171] In some embodiments, the CRISPR enzyme comprises a Rec2 or
HD2 truncation. The Rec2 or HD2 domains are known in Sp Cas9 from
the crystal structure provided by Nishimasu et al. and the herein
cited materials; corresponding domains are envisaged in orthologs.
Such mutants may be advantageous where there is a desire to reduce
the package size as this can assist with delivery, especially with
the larger Cas9s such as Sp Cas9. It will be appreciated that
truncation may include removal of the domain, in some embodiments.
In some embodiments, the truncation includes replacement with a
different amino acid sequence, for example a linker. In some
embodiments, the linker is branched or otherwise allows for
tethering of the DD and/or a functional domain to the CRISPR
enzyme. Functional domains are discussed further herein. HD2, the
Helical Domain 2, is dispensable (meaning that at least 10%
functional CRISPR enzyme activity is retained, preferably at least
30% and most preferably at least 50% functional CRISPR enzyme
activity is retained in the truncated CRISPR enzyme). An exemplary
DNA sequence encoding it in Sp Cas9 is provided below and suitable
equivalents will be readily apparent in orthologs of Sp via
sequence comparison, using programs such as BLAST. HD2 domain in Sp
Cas9:
TABLE-US-00001 (SEQ ID NO: 42)
CTGAACCCCGACAACAGCGACGTGGACAAGCTGTTCATCCAGCTGGTGCA
GACCTACAACCAGCTGTTCGAGGAAAACCCCATCAACGCCAGCGGCGTGG
ACGCCAAGGCCATCCTGTCTGCCAGACTGAGCAAGAGCAGACGGCTGGAA
AATCTGATCGCCCAGCTGCCCGGCGAGAAGAAGAATGGCCTGTTCGGAAA
CCTGATTGCCCTGAGCCTGGGCCTGACCCCCAACTTCAAGAGCAACTTCG
ACCTGGCCGAGGATGCCAAACTGCAGCTGAGCAAGGACACCTACGACGAC
GACCTGGACAACCTGCTGGCCCAGATCGGCGACCAGTACGCCGACCTGTT
TCTGGCCGCCAAGAACCTGTCCGACGCCATCCTGCTGAGCGACATCCTGA
GAGTGAACACCGAG
[0172] In an aspect the invention involves a split Cas9 system,
providing an additional level of control on top of the DD-Cas9 of
the present invention, as in Zetsche et al (Zetsche et al., "A
split-Cas9 architecture for inducible genome editing and
transcription modulation," Nature Biotechnology 33:139-142,
DOI:10.1038/nbt.3149 (Published online 2 Feb. 2015)). For example,
the invention can provide a non-naturally occurring or engineered
inducible CRISPR-Cas system, comprising: a first CRISPR enzyme
fusion construct attached to a first half of an inducible dimer and
a second CRISPR enzyme fusion construct attached to a second half
of the inducible dimer,
[0173] wherein the first CRISPR enzyme fusion construct comprises a
first part of a CRISPR enzyme of the present invention and is
operably linked to one or more nuclear localization signals,
[0174] wherein the second CRISPR enzyme fusion construct comprises
a second part of a CRISPR enzyme of the present invention is
operably linked to one or more nuclear export signals,
[0175] wherein contact with an inducer energy source brings the
first and second halves of the inducible dimer together,
[0176] wherein bringing the first and second halves of the
inducible dimer together brings the first and second parts of the
CRISPR enzyme together and thereby allows the first and second
CRISPR enzyme fusion constructs to constitute a functional
CRISPR-Cas system,
[0177] wherein the CRISPR-Cas system comprises a guide RNA (sgRNA)
comprising a guide sequence capable of hybridizing to a target
sequence in a genomic locus of interest in a cell, and
[0178] wherein the functional CRISPR-Cas system binds to the target
sequence and, optionally, edits the genomic locus to alter gene
expression.
[0179] In an aspect of the invention in the inducible CRISPR-Cas
system, the CRISPR enzyme comprises two parts of a split CRISPR
enzyme. With regard to the inducible system, the terms CRISPR
enzyme and split CRISPR enzyme may be used interchangeably. In an
aspect of the invention in the inducible CRISPR-Cas system, the
inducible dimer is or comprises or consists essentially of or
consists of an inducible heterodimer. In an aspect, in inducible
CRISPR-Cas system, the first half or a first portion or a first
fragment of the inducible heterodimer is or comprises or consists
of or consists essentially of an FKBP, optionally FKBP12. In an
aspect of the invention, in the inducible CRISPR-Cas system, the
second half or a second portion or a second fragment of the
inducible heterodimer is or comprises or consists of or consists
essentially of FRB. In an aspect of the invention, in the inducible
CRISPR-Cas system, the arrangement of the first CRISPR enzyme
fusion construct is or comprises or consists of or consists
essentially of N-terminal Cas9 part-FRB-NES. In an aspect of the
invention, in the inducible CRISPR-Cas system, the arrangement of
the first CRISPR enzyme fusion construct is or comprises or
consists of or consists essentially of NES-N-terminal Cas9
part-FRB-NES. In an aspect of the invention, in the inducible
CRISPR-Cas system, the arrangement of the second CRISPR enzyme
fusion construct is or comprises or consists essentially of or
consists of C-terminal Cas9 part-FKBP-NLS. In an aspect the
invention provides in the inducible CRISPR-Cas system, the
arrangement of the second CRISPR enzyme fusion construct is or
comprises or consists of or consists essentially of NLS-C-terminal
Cas9 part-FKBP-NLS. In an aspect, in inducible CRISPR-Cas system
there can be a linker that separates the Cas9 part from the half or
portion or fragment of the inducible dimer. In an aspect, in the
inducible CRISPR-Cas system, the inducer energy source is or
comprises or consists essentially of or consists of rapamycin. In
an aspect, in inducible CRISPR-Cas system, the inducible dimer is
an inducible homodimer. In an aspect, in inducible CRISPR-Cas
system, the CRISPR enzyme is Cas9, e.g., SpCas9 or SaCas9.
[0180] Inducer for Split Cas9: An inducer energy source may be
considered to be simply an inducer or a dimerizing agent. The term
`inducer energy source` is used herein throughout for consistency.
The inducer energy source (or inducer) acts to reconstitute the
Cas9. In some embodiments, the inducer energy source brings the two
parts of the Cas9 together through the action of the two halves of
the inducible dimer. The two halves of the inducible dimer
therefore are brought tougher in the presence of the inducer energy
source. The two halves of the dimer will not form into the dimer
(dimerize) without the inducer energy source. Thus, the two halves
of the inducible dimer cooperate with the inducer energy source to
dimerize the dimer. This in turn reconstitutes the Cas9 by bringing
the first and second parts of the Cas9 together. Suitable examples
include rapamycin.
[0181] Split Position: In an aspect in inducible CRISPR-Cas system,
the Cas9 is split into two parts at any one of the following split
points, according or with reference to SpCas9: a split position
between 202A/2035; a split position between 255F/256D; a split
position between 310E/311I; a split position between 534R/535K; a
split position between 572E/573C; a split position between
7135/714G; a split position between 1003L/104E; a split position
between 1054G/1055E; a split position between 1114N/1115S; a split
position between 1152K/1153S; a split position between 1245K/1246G;
or a split between 1098 and 1099. Cas9 can be split into two
distinct fragments, which reconstitute a functional full-length
Cas9 nuclease when brought back together using chemical induction.
The split Cas9 architecture will be useful for a variety of
applications. For example, split Cas9 may enable genetic strategies
for restricting Cas9 activity to intersectional cell populations by
putting each fragment under a different tissue specific promoter.
Additionally, different chemically inducible dimerization domains
such as APA and gibberellin may also be employed. The split
position or location is the point at which the first part of the
Cas9 enzyme is separated from the second part. In some embodiments,
the first will comprise or encode amino acids 1 to X, whilst the
second part will comprise or encode amino acids X+1 to the end. In
this example, the numbering is contiguous, but this may not always
be necessary as amino acids (or the nucleotides encoding them)
could be trimmed from the end of either of the split ends, provided
that sufficient DNA binding activity and, if required, DNA nickase
or cleavage activity is retained, for example at least 40%, 50%,
60%, 70%, 80%, 90% or 95% activity compared to wildtype Cas9.
[0182] The exemplary numbering provided herein may be in reference
to the wildtype protein, preferably the wildtype SpCas9 protein.
However, it is envisaged that mutants of the wildtype SpCas9
protein can be used. For example, in the crystal data paper itself,
a deadCas9 was used and these are preferred in some embodiments,
see the discussion elsewhere herein. The numbering may also not
follow exactly the Sp Cas9 numbering as, for instance, some N- or
C-terminal truncations or deletions may be used, but this can be
addressed suing standard sequence alignment tools. Orthologs are
also preferred as a sequence alignment tool. Thus, the split
position may be selected using ordinary skill in the art, for
instance based on the crystal data provided in the herein cited
materials. A number of split positions in SpCas9, which
reconstitute Cas9 with inducible dimerization domains, include as
tabulated below (showing Amino Acid position of split in Sp Cas9
(1368 a.a. in total)):
TABLE-US-00002 Fusion Side Structure Domain 202A/203S Outside loop
Rec 2 255F/256D Outside loop Rec 2 310E/311I Outside loop Rec 1
534R/535K Outside loop Rec 1 572E/573C Unstructured Rec 1 713S/714G
Unstructured Rec 1 1003L/104E Unstructured RuvC3 1054G/1055E
Unstructured RuvC3 1114N/1115S Unstructured PI 1152K/1153S Outside
loop PI 1245K/1246G Unstructured PI
[0183] The following split positions may also be advantageously
employed (Amino Acid position of split in Sp Cas9 (1368 a.a. in
total)):
TABLE-US-00003 Amino Acid position Split in Loop (L) Split of Sp
Cas9 (1368 a.a. or Unstructured number in total) Domain Region
(UR)? 1 203 Rec 2 L 2 256 Rec 2 L 3 311 Rec 1 L 4 535 Rec 1 L 5 573
Rec 1 UR 6 714 Rec 1 UR 7 1004 RuvC3 UR 8 1055 RuvC3 UR 9 1115 PI
UR 10 1153 PI L 11 1246 PI UR
Identifying potential split sides is most simply done with the help
of a crystal structure. For Sp mutants, it should be readily
apparent what the corresponding position for, for example, a
sequence alignment. For non-Sp enzymes one can use the crystal
structure of an ortholog if a relatively high degree of homology
exists between the ortholog and the intended Cas9.
[0184] The split position may be located within a region or loop.
Preferably, the split position occurs where an interruption of the
amino acid sequence does not result in the partial or full
destruction of a structural feature (e.g. alpha-helixes or
beta-sheets). Unstructured regions (regions that did not show up in
the crystal structure because these regions are not structured
enough to be "frozen" in a crystal) are often preferred options.
Splits in all unstructured regions that are exposed on the surface
of SpCas9 are envisioned in the practice of the invention. The
positions within the unstructured regions or outside loops may not
need to be exactly the numbers provided above, but may vary by, for
example 1, 2, 3, 4, 5, 6, 7, 8, 9, or even 10 amino acids either
side of the position given above, depending on the size of the
loop, so long as the split position still falls within an
unstructured region of outside loop.
[0185] A split in an outside loop of the Rec 2 domain is preferred
in some embodiments. In other embodiments, a split in an outside
loop of Rec 1 is preferred. In other embodiments, a split in an
outside loop of PI is preferred. In other embodiments, a split in
an unstructured region of Rec 1 is preferred. In other embodiments,
a split in an unstructured region of RuvC3 is preferred. In other
embodiments, a split in an unstructured region of PI is
preferred.
[0186] Splits 4, 5 and 6 in Table 2 above are beneficial in one
aspect, in that there is some advantage to keeping the two parts
(either side of the split) roughly the same length for packing
purposes. For example, it is thought to be easier to maintain
stoichiometry between both pieces when the transcripts are about
the same size.
[0187] The N- and C-term pieces of human codon-optimized S.
pyogenes Cas9 may be fused to FRB and FKBP dimerization domains,
respectively. This arrangement is preferred. They may be switched
over (i.e. N-term to FKBP and C-term to FRB), this arrangement
worked as well but there is a suggestion that this switched
arrangement brings the two parts of the Cas9 further apart.
[0188] Linkers such as (GGGGS).sub.3 (SEQ ID NO: 27) are preferably
used herein to separate the Cas9 fragment from the dimerization
domain. (GGGGS).sub.3 (SEQ ID NO: 27) is preferable because it is a
relatively long linker (15 amino acids). The glycine residues are
the most flexible and the serine residues enhance the chance that
the linker is on the outside of the protein. (GGGGS).sub.6 (SEQ ID
NO: 31) (GGGGS).sub.9 (SEQ ID NO: 32) or (GGGGS).sub.12 (SEQ ID NO:
33) may preferably be used as alternatives. Other preferred
alternatives are (GGGGS).sub.1 (SEQ ID NO: 34), (GGGGS).sub.2 (SEQ
ID NO: 35), (GGGGS).sub.4 (SEQ ID NO: 36), (GGGGS).sub.5 (SEQ ID
NO: 37), (GGGGS).sub.7 (SEQ ID NO: 38), (GGGGS).sub.8 (SEQ ID NO:
39), (GGGGS).sub.10 (SEQ ID NO: 40), or (GGGGS).sub.11 (SEQ ID NO:
41). For example, (GGGGS).sub.3 (SEQ ID NO: 27) may be used between
the N-term Cas9 fragment and FRB. Such a linker may also be used
between FKB and the C-term Cas9 fragment. Alternative linkers are
available, but highly flexible linkers are thought to work best to
allow for maximum opportunity for the 2 parts of the Cas9 to come
together and thus reconstitute Cas9 activity. One alternative is
that the NLS of nucleoplasmin can be used as a linker. A linker,
such as any of the linkers discussed herein, can also be used
between the Cas9 and any functional domain. Again, a (GGGGS).sub.3
linker (SEQ ID NO: 27) may be used here (or the 6, 9, or 12 repeat
versions therefore) or the NLS of nucleoplasmin can be used as a
linker between Cas9 and the functional domain.
[0189] In some embodiments, the FRB/FKBP system is preferred.
However, alternatives to the FRB/FKBP system are envisaged. For
example the ABA and gibberellin system. Accordingly, preferred
examples of the FKBP family are any one of the following inducible
systems. FKBP which dimerizes with CalcineurinA (CNA), in the
presence of FK506; FKBP which dimerizes with CyP-Fas, in the
presence of FKCsA; FKBP which dimerizes with FRB, in the presence
of Rapamycin; GyrB which dimerizes with GryB, in the presence of
Coumermycin; GAI which dimerizes with GID1, in the presence of
Gibberellin; or Snap-tag which dimerizes with HaloTag, in the
presence of HaXS. Alternatives within the FKBP family itself are
also preferred. For example, FKBP, which homo-dimerizes (i.e. one
FKBP dimerizes with another FKBP) in the presence of FK1012. Thus,
also provided is a non-naturally occurring or engineered inducible
CRISPR-Cas system, comprising: a first CRISPR enzyme fusion
construct attached to a first half of an inducible homodimer and a
second CRISPR enzyme fusion construct attached to a second half of
the inducible homodimer, wherein the first CRISPR enzyme fusion
construct is operably linked to one or more nuclear localization
signals, wherein the second CRISPR enzyme fusion construct is
operably linked to a (optionally one or more) nuclear export
signal(s), wherein contact with an inducer energy source brings the
first and second halves of the inducible homodimer together,
wherein bringing the first and second halves of the inducible
homodimer together allows the first and second CRISPR enzyme fusion
constructs to constitute a functional CRISPR-Cas system, wherein
the CRISPR-Cas system comprises a guide RNA (sgRNA) comprising a
guide sequence capable of hybridizing to a target sequence in a
genomic locus of interest in a cell, and wherein the functional
CRISPR-Cas system binds to the target sequence and, optionally,
edits the genomic locus to alter gene expression.
[0190] In one embodiment, the homodimer is preferably FKBP and the
inducer energy source is preferably FK1012. In another embodiment,
the homodimer is preferably GryB and the inducer energy source is
preferably Coumermycin. In another embodiment, the homodimer is
preferably ABA and the inducer energy source is preferably
Gibberellin. In other embodiments, the dimer is a heterodimer.
Preferred examples of heterodimers are any one of the following
inducible systems: FKBP which dimerizes with CalcineurinA (CNA), in
the presence of FK506; FKBP which dimerizes with CyP-Fas, in the
presence of FKCsA; FKBP which dimerizes with FRB, in the presence
of Rapamycin, in the presence of Coumermycin; GAI which dimerizes
with GID1, in the presence of Gibberellin; or Snap-tag which
dimerizes with HaloTag, in the presence of HaXS. FKBP/FRB is
advantageous as it is well characterized and both domains are
sufficiently small (<100 amino acids) to assist with packaging.
Furthermore, rapamycin has been used for a long time and side
effects are well understood. Large dimerization domains (>300
aa) should work too but may require longer linkers to make enable
Cas9 reconstitution.
[0191] Teachings in Paulmurugan and Gambhir (Cancer Res, Aug. 15,
2005 65; 7413) may be used in combination with herein teachings to
exemplify the FRB/FKBP/Rapamycin system in the practice of the
invention; see also Crabtree et al. (Chemistry & Biology 13,
99-107, January 2006).
[0192] An inducer energy source may be considered to be simply an
inducer or a dimerizing agent. The term `inducer energy source` is
used herein throughout for consistency. The inducer energy source
(or inducer) acts to reconstitute the Cas9. In some embodiments,
the inducer energy source brings the two parts of the Cas9 together
through the action of the two halves of the inducible dimer. The
two halves of the inducible dimer therefore are brought tougher in
the presence of the inducer energy source. The two halves of the
dimer will not form into the dimer (dimerize) without the inducer
energy source.
[0193] Thus, the two halves of the inducible dimer cooperate with
the inducer energy source to dimerize the dimer. This in turn
reconstitutes the Cas9 by bringing the first and second parts of
the Cas9 together.
[0194] A single vector can be used. An expression cassette
(plasmid) was constructed as follows. The split Cas9 construct was
based on a first CRISPR enzyme fusion construct, flanked by NLSs,
with FKBP fused to C terminal part of the split Cas9 via a GlySer
linker; and a second CRISPR enzyme fusion construct, flanked by
NESs, with FRB fused with the N terminal part of the split Cas9 via
a GlySer linker. To separate the first and second CRISPR enzyme
fusion constructs, P2A was used splitting on transcription. The
Split Cas9s showed indel formation similar to wildtype in the
presence of rapamycin, but markedly lower indel formation than the
wildtype in the absence of rapamycin. Accordingly, a single vector
is provided. The vector comprises: a first CRISPR enzyme fusion
construct attached to a first half of an inducible dimer and a
second CRISPR enzyme fusion construct attached to a second half of
the inducible dimer, wherein the first CRISPR enzyme fusion
construct is operably linked to one or more nuclear localization
signals, wherein the second CRISPR enzyme fusion construct is
operably linked to one or more nuclear export signals, wherein
contact with an inducer energy source brings the first and second
halves of the inducible heterodimer together, wherein bringing the
first and second halves of the inducible heterodimer together
allows the first and second CRISPR enzyme fusion constructs to
constitute a functional CRISPR-Cas system, wherein the CRISPR-Cas
system comprises a guide RNA (sgRNA) comprising a guide sequence
capable of hybridizing to a target sequence in a genomic locus of
interest in a cell, and wherein the functional CRISPR-Cas system
binds to the target sequence and, optionally, edits the genomic
locus to alter gene expression. These elements are preferably
provided on a single construct, for example an expression
cassette.
[0195] The first CRISPR enzyme fusion construct is preferably
flanked by at least one nuclear localization signal at each end.
The second CRISPR enzyme fusion construct is preferably flanked by
at least one nuclear export signal at each end.
[0196] Applicants have determined new split sites for SaCas9.
TABLE-US-00004 Fusion Side Structure Domain 430/431 Unstructured
Between REC and NUC lobes (between 3'/C' loop terminal end of Rec
Domain and 5'/N' terminal end of RuvCII domain). 739/740
Unstructured RuvCIII domain region
Table showing Amino Acid position of split in Sa Cas9 (1053 a.a. in
total)
[0197] In some embodiments, with any Cas9, the split is preferably
positioned in an unstructured loop or an unstructured region.
[0198] In some embodiments, with split SaCas9, the split is
preferably positioned at or around amino acid position 430 or 431,
in particular between 430 and 431.
[0199] In some embodiments, with split SaCas9, the split is
preferably positioned at or around amino acid position 739 or 740,
in particular between 739 and 740.
[0200] With split SaCas9, a certain amount of variation should be
tolerated on each side of each split. For example, for split point
1: -4 to +2 amino acid positions is ideal. In some embodiments, the
split is positioned at (ideally C' terminal to) amino acid
positions 426, 427, 428, 429 or 430. In some embodiments, the split
is positioned at (ideally C' terminal to) amino acid positions 431,
432 or even 433. In some embodiments, the split is positioned at
(ideally C' terminal to) amino acid positions 426-433.
[0201] Without being bound by theory, it is understood that any
further towards the N' terminal than -4 and the split position gets
too close to the sgRNA. Further towards the C' terminal than +2 and
the split position gets too close to an alpha-helix.
[0202] For split point 2: the split point at 739-740 may be moved
within -7 to +4 amino acid positions. In some embodiments, the
split is positioned at (ideally C' terminal to) amino acid
positions 732, 733, 734, 735, 736, 737, 738, or 739. In some
embodiments, the split is positioned at (ideally C' terminal to)
amino acid positions 740, 471, 742, 743, 744 or even 745. In some
embodiments, the split is positioned at (ideally C' terminal to)
amino acid positions 732-745.
[0203] Without being bound by theory, it is understood that split 2
is located in the middle of an unstructured region flanked by
alpha-helixes.
[0204] Corresponding Positions in Other Orthologues are Also
Envisaged
[0205] The promoter used for SaCas9 splits is CBh and bGHpA is the
polyA signal in all constructs cloned so far and are planning to
clone. However, we know that CMV, EF1alpha and EFS (minimal EF1a
promoter) work well for SpCas9 and will also work for SaCas9.
[0206] In an aspect, the promoter used for SaCas9 splits is CBh. In
other aspects, CMV, EF1alpha and EFS (minimal EF1.alpha. promoter)
may be used as promoters. In an aspect, a polyA tail such as bGHpA
may be used.
[0207] Also provided is a split SaCas9 with specific split points.
In an aspect the invention provides a non-naturally occurring or
engineered inducible CRISPR-Cas system, comprising: a first CRISPR
enzyme fusion construct attached to a first half of an inducible
dimer and a second CRISPR enzyme fusion construct attached to a
second half of the inducible dimer,
[0208] wherein the first CRISPR enzyme fusion construct comprises a
first part of a CRISPR enzyme,
[0209] wherein the second CRISPR enzyme fusion construct comprises
a second part of a CRISPR enzyme,
[0210] wherein contact with an inducer energy source brings the
first and second halves of the inducible dimer together,
[0211] wherein bringing the first and second halves of the
inducible dimer together brings the first and second parts of the
CRISPR enzyme together and thereby allows the first and second
CRISPR enzyme fusion constructs to constitute a functional
CRISPR-Cas system,
[0212] wherein the CRISPR-Cas system comprises a guide RNA (sgRNA)
comprising a guide sequence capable of hybridizing to a target
sequence in a genomic locus of interest in a cell, and
[0213] wherein the functional CRISPR-Cas system binds to the target
sequence and, optionally, edits the genomic locus to alter gene
expression, and
[0214] wherein the two parts of the Cas9 are split between amino
acid positions amino acid positions 426-433 or amino acid positions
732-745, or positions corresponding thereto. In an aspect, the Cas9
is SaCas9. In an aspect, the Cas9 is an ortholog of SaCas9. In an
aspect, the first CRISPR enzyme fusion construct comprises a first
part of a CRISPR enzyme and is operably linked to one or more,
preferably two or more nuclear localization signals (NLS). In an
aspect, the first CRISPR enzyme fusion construct comprises a first
part of a CRISPR enzyme and is operably linked to one or more,
preferably two or more nuclear export signals (NES). In an aspect,
the second CRISPR enzyme fusion construct comprises a second part
of a CRISPR enzyme and is operably linked to one or more,
preferably two or more nuclear localization signals. In an aspect,
the second CRISPR enzyme fusion construct comprises a second part
of a CRISPR enzyme and is operably linked to one or more,
preferably two or more nuclear export signals. In an aspect, the
first CRISPR enzyme fusion construct comprises a first part of a
CRISPR enzyme and is operably linked to one or more, preferably two
or more nuclear localization signals and the second CRISPR enzyme
fusion construct comprises a second part of a CRISPR enzyme and is
operably linked to one or more, preferably two or more nuclear
export signals. In an aspect, the first CRISPR enzyme fusion
construct comprises a first part of a CRISPR enzyme and is operably
linked to one or more, preferably two or more nuclear export
signals and the second CRISPR enzyme fusion construct comprises a
second part of a CRISPR enzyme and is operably linked to one or
more, preferably two or more nuclear localization signals.
[0215] In an aspect, two NLS are operably linked to one part, and
one NES operably is operably linked to the other part. In a further
aspect, one or two NLS are operably linked to one part, and one or
two NES operably are also operably linked to the other part.
[0216] In addition as to Split Cas9, in an aspect, in the inducible
CRISPR-Cas system, one or more functional domains, as discussed
herein are associated, with one or both parts of the Cas9 enzyme.
For example, the functional domains may optionally include a
transcriptional activator, a transcriptional or a nuclease such as
a Fok1 nuclease. Further examples are provided herein. In an
aspect, in the inducible CRISPR-Cas system, the functional
CRISPR-Cas system binds to the target sequence and the enzyme is a
deadCas9, optionally having a diminished nuclease activity of at
least 97%, or 100% (or no more than 3% and advantageously 0%
nuclease activity) as compared with the CRISPR enzyme not having
the at least one mutation. In an aspect, in the inducible
CRISPR-Cas system, the deadCas9 (CRISPR enzyme) comprises two or
more mutations wherein two or more of D10, E762, H840, N854, N863,
or D986 according to SpCas9 protein or any corresponding ortholog
or N580 according to SaCas9 protein are mutated, or the CRISPR
enzyme comprises at least one mutation, e.g., wherein at least H840
is mutated.
[0217] In some embodiments, the one or more DDs may be used. The or
each DD may be associated with one of the two parts of the split
CRISPR enzyme.
[0218] Where one DD is used, it is preferred in some embodiments,
that the NES is associated with the same part of the split CRISPR
enzyme as the DD. This may help to ensure cytoplasmic location of
the DD.
[0219] Where more than one DD is used, it is preferred in some
embodiments, that the NES is associated with the same part of the
split CRISPR enzyme as at least one of the DDs. Again, this may
help to ensure cytoplasmic location of the DD.
[0220] In some embodiments, where one part of the split CRISPR
enzyme comprises two or more DDs, there may be one DD associated
with the N-terminal of the split CRISPR enzyme and one DD
associated with the C-terminal of the CRISPR enzyme.
[0221] In some embodiments, two DDs may be used. Both may be
associated with the same part of the split CRISPR enzyme, or one DD
may be associated with one part of the split CRISPR enzyme (N- or
C-terminal) and the other DD may be associated with the other part
of the split CRISPR enzyme (N- or C-terminal).
[0222] In some embodiments, three DDs may be used. All three may be
associated with the same part of the split CRISPR enzyme (N- or
C-terminal or a mixture), or one DD may be associated with one part
of the split CRISPR enzyme (N- or C-terminal) and the other two DDs
may be associated with the other part of the split CRISPR enzyme
(N- or C-terminal or a mixture).
[0223] In some embodiments, four DDs may be used. All four may be
associated with the same part of the split CRISPR enzyme (N- or
C-terminal or a mixture), or one DD may be associated with one part
of the split CRISPR enzyme (N- or C-terminal) and the other three
DDs may be associated with the other part of the split CRISPR
enzyme (N- or C-terminal or a mixture).
[0224] In some embodiments, four DDs may be used and two DDs may be
associated with one part of the split CRISPR enzyme (N- or
C-terminal or a mixture) and the other two DDs may be associated
with the other part of the split CRISPR enzyme (N- or C-terminal or
a mixture). In some embodiments using four DDs, one DD may be
associated with each end of the two split CRISPR enzyme parts. This
paired approach is preferred as it allows one DD at each end of
both parts of the split CRISPR enzyme.
[0225] As such, in some embodiments, one DD is associated with the
N-terminal of the first part of the split CRISPR enzyme and one DD
is associated with the C-terminal of the first part of the CRISPR
enzyme; and one DD is associated with the N-terminal of the second
part of the split CRISPR enzyme and one DD is associated with the
C-terminal of the second part of the CRISPR enzyme. The invention
further comprehends and an aspect of the invention provides, a
polynucleotide encoding the inducible CRISPR-Cas system as herein
discussed.
[0226] One guide with a first aptamer/RNA-binding protein pair can
be linked or fused to an activator, while a second guide with a
second aptamer/RNA-binding protein pair can be linked or fused to a
repressor. The guides are for different targets (loci), so this
allows one gene to be activated and one repressed. For example, the
following schematic shows such an approach:
Guide 1--MS2 aptamer - - - MS2 RNA-binding protein - - - VP64
activator; and Guide 2--PP7 aptamer - - - PP7 RNA-binding protein -
- - SID4x repressor.
[0227] The present invention also relates to orthogonal PP7/MS2
gene targeting. In this example, sgRNA targeting different loci are
modified with distinct RNA loops in order to recruit MS2-VP64 or
PP7-SID4X, which activate and repress their target loci,
respectively. PP7 is the RNA-binding coat protein of the
bacteriophage Pseudomonas. Like MS2, it binds a specific RNA
sequence and secondary structure. The PP7 RNA-recognition motif is
distinct from that of MS2. Consequently, PP7 and MS2 can be
multiplexed to mediate distinct effects at different genomic loci
simultaneously. For example, an sgRNA targeting locus A can be
modified with MS2 loops, recruiting MS2-VP64 activators, while
another sgRNA targeting locus B can be modified with PP7 loops,
recruiting PP7-SID4X repressor domains. In the same cell, dCas9 can
thus mediate orthogonal, locus-specific modifications. This
principle can be extended to incorporate other orthogonal
RNA-binding proteins such as Q-beta.
[0228] An alternative option for orthogonal repression includes
incorporating non-coding RNA loops with transactive repressive
function into the guide (either at similar positions to the MS2/PP7
loops integrated into the guide or at the 3' terminus of the
guide). For instance, guides were designed with non-coding (but
known to be repressive) RNA loops (e.g., using the Alu repressor
(in RNA) that interferes with RNA polymerase II in mammalian
cells). The Alu RNA sequence was located: in place of the MS2 RNA
sequences as used herein (e.g., at tetraloop and/or stem loop 2);
and/or at 3' terminus of the guide. This gives possible
combinations of MS2, PP7 or Alu at the tetraloop and/or stemloop 2
positions, as well as, optionally, addition of Alu at the 3' end of
the guide (with or without a linker).
[0229] The use of two different aptamers (each associated with a
distinct RNA) allows an activator-adaptor protein fusion and a
repressor-adaptor protein fusion to be used, with different guides,
to activate expression of one gene, while repressing another. They,
along with their different guides can be administered together, or
substantially together, in a multiplexed approach. A large number
of such modified guides can be used all at the same time, for
example 10 or 20 or 30 and so forth, while only one (or at least a
minimal number) of Cas9s to be delivered, as a comparatively small
number of Cas9s can be used with a large number modified guides.
The adaptor protein may be associated (preferably linked or fused
to) one or more activators or one or more repressors. For example,
the adaptor protein may be associated with a first activator and a
second activator. The first and second activators may be the same,
but they are preferably different activators. For example, one
might be VP64, while the other might be p65, although these are
just examples and other transcriptional activators are envisaged.
Three or more or even four or more activators (or repressors) may
be used, but package size may limit the number being higher than 5
different functional domains. Linkers are preferably used, over a
direct fusion to the adaptor protein, where two or more functional
domains are associated with the adaptor protein. Suitable linkers
might include the GlySer linker.
[0230] It is also envisaged that the enzyme-guide complex as a
whole may be associated with two or more functional domains. For
example, there may be two or more functional domains associated
with the enzyme, or there may be two or more functional domains
associated with the guide (via one or more adaptor proteins), or
there may be one or more functional domains associated with the
enzyme and one or more functional domains associated with the guide
(via one or more adaptor proteins).
[0231] The fusion between the adaptor protein and the activator or
repressor may include a linker. For example, GlySer linkers GGGS
(SEQ ID NO: 28) can be used. They can be used in repeats of 3
((GGGGS).sub.3 (SEQ ID NO: 27)) or 6 (SEQ ID NO: 31), 9 (SEQ ID NO:
32) or even 12 (SEQ ID NO: 33) or more, to provide suitable
lengths, as required. Linkers can be used between the RNA-binding
protein and the functional domain (activator or repressor), or
between the CRISPR Enzyme (Cas9) and the functional domain
(activator or repressor). The linkers the user to engineer
appropriate amounts of "mechanical flexibility".
[0232] The invention comprehends a DD-CRISPR Cas complex comprising
a DD-CRISPR enzyme and a guide RNA (sgRNA), wherein the DD-CRISPR
enzyme comprises at least one mutation, such that the DD-CRISPR
enzyme has no more than 5% of the nuclease activity of the CRISPR
enzyme not having the at least one mutation and, optional, at least
one or more nuclear localization sequences; the guide RNA (sgRNA)
comprises a guide sequence capable of hybridizing to a target
sequence in a genomic locus of interest in a cell; and wherein: the
DD-CRISPR enzyme is associated with two or more functional domains;
or at least one loop of the sgRNA is modified by the insertion of
distinct RNA sequence(s) that bind to one or more adaptor proteins,
and wherein the adaptor protein is associated with two or more
functional domains; or the DD-CRISPR enzyme is associated with one
or more functional domains and at least one loop of the sgRNA is
modified by the insertion of distinct RNA sequence(s) that bind to
one or more adaptor proteins, and wherein the adaptor protein is
associated with one or more functional domains. The invention
comprehends the use of modified guides such as in Konermann et al.,
"Genome-scale transcription activation by an engineered CRISPR-Cas9
complex," doi:10.1038/nature14136, incorporated herein by
reference, or PCT/US14/70175, filed Dec. 12, 2014.
[0233] In some embodiments, one or more functional domains are
associated with an adaptor protein, for example as used with the
modified guides of Konermann et al. (Nature 517, 583-588, 29 Jan.
2015).
[0234] For the purposes of the following discussion, reference to a
functional domain could be a functional domain associated with the
CRISPR enzyme or a functional domain associated with the adaptor
protein.
[0235] In some embodiments, the one or more functional domains is
an NLS (Nuclear Localization Sequence) or an NES (Nuclear Export
Signal). In some embodiments, the one or more functional domains is
a transcriptional activation domain comprises VP64, p65, MyoD1,
HSF1, RTA, SET7/9 and a histone acetyltransferase. Other references
herein to activation (or activator) domains in respect of those
associated with the CRISPR enzyme include any known transcriptional
activation domain and specifically VP64, p65, MyoD1, HSF1, RTA,
SET7/9 or a histone acetyltransferase.
[0236] In some embodiments, the one or more functional domains is a
transcriptional repressor domain. In some embodiments, the
transcriptional repressor domain is a KRAB domain. In some
embodiments, the transcriptional repressor domain is a NuE domain,
NcoR domain, SID domain or a SID4X domain.
[0237] In some embodiments, the one or more functional domains have
one or more activities comprising methylase activity, demethylase
activity, transcription activation activity, transcription
repression activity, transcription release factor activity, histone
modification activity, RNA cleavage activity, DNA cleavage
activity, DNA integration activity or nucleic acid binding
activity.
[0238] Histone modifying domains are also preferred in some
embodiments. Exemplary histone modifying domains are discussed
below. Transposase domains, HR (Homologous Recombination) machinery
domains, recombinase domains, and/or integrase domains are also
preferred as the present functional domains. In some embodiments,
DNA integration activity includes HR machinery domains, integrase
domains, recombinase domains and/or transposase domains. Histone
acetyltransferases are preferred in some embodiments.
[0239] In some embodiments, the DNA cleavage activity is due to a
nuclease. In some embodiments, the nuclease comprises a Fok1
nuclease.
[0240] In some embodiments, the one or more functional domains is
attached to the CRISPR enzyme so that upon binding to the sgRNA and
target the functional domain is in a spatial orientation allowing
for the functional domain to function in its attributed
function.
[0241] In some embodiments, the one or more functional domains is
attached to the adaptor protein so that upon binding of the CRISPR
enzyme to the sgRNA and target, the functional domain is in a
spatial orientation allowing for the functional domain to function
in its attributed function.
[0242] In an aspect the invention provides a composition as herein
discussed wherein the one or more functional domains is attached to
the CRISPR enzyme or adaptor protein via a linker, optionally a
GlySer linker, as discussed herein.
[0243] Endogenous transcriptional repression is often mediated by
chromatin modifying enzymes such as histone methyltransferases
(HMTs) and deacetylases (HDACs). Repressive histone effector
domains are known and an exemplary list is provided below. In the
exemplary table, preference was given to proteins and functional
truncations of small size to facilitate efficient viral packaging
(for instance via AAV). In general, however, the domains may
include HDACs, histone methyltransferases (HMTs), and histone
acetyltransferase (HAT) inhibitors, as well as HDAC and HMT
recruiting proteins. The functional domain may be or include, in
some embodiments, HDAC Effector Domains, HDAC Recruiter Effector
Domains, Histone Methyltransferase (HMT) Effector Domains, Histone
Methyltransferase (HMT) Recruiter Effector Domains, or Histone
Acetyltransferase Inhibitor Effector Domains.
TABLE-US-00005 HDAC Effector Domains Complex Full Selected Final
Substrate Modification size truncation size Catalytic Subtype Name
(if known) (if known) Organism (aa) (aa) (aa) domain HDAC I HDAC8
-- -- X. 325 1-325 325 1-272: HDAC laevis HDAC I RPD3 -- -- S. 433
19-340 322 19-331: HDAC cerevisiae (Vannier) HDAC IV MesoLo4 -- --
M. loti 300 1-300 300 -- (Gregoretti) HDAC IV HDAC11 -- -- H. 347
1-347 347 14-326: HDAC sapiens (Gao) HD2 HDT1 -- -- A. 245 1-211
211 -- thaliana (Wu) SIRT I SIRT3 H3K9Ac -- H. 399 143-399 257
126-382: SIRT H4K16Ac sapiens (Scher) H3K56Ac SIRT I HST2 -- -- C.
331 1-331 331 -- albicans (Hnisz) SIRT I CobB -- -- E. coli 242
1-242 242 -- (K12) (Landry) SIRT I HST2 -- -- S. 357 8-298 291 --
cerevisiae (Wilson) SIRT III SIRT5 H4K8Ac -- H. 310 37-310 274
41-309: SIRT H4K16Ac sapiens (Gertz) SIRT III Sir2A -- -- P. 273
1-273 273 19-273: SIRT falciparum (Zhu) SIRT IV SIRT6 H3K9Ac -- H.
355 1-289 289 35-274: SIRT H3K56Ac sapiens (Tennen)
[0244] Accordingly, the repressor domains of the present invention
may be selected from histone methyltransferases (HMTs), histone
deacetylases (HDACs), histone acetyltransferase (HAT) inhibitors,
as well as HDAC and HMT recruiting proteins.
[0245] The HDAC domain may be any of those in the table above,
namely: HDAC8, RPD3, MesoLo4, HDAC11, HDT1, SIRT3, HST2, CobB,
HST2, SIRT5, Sir2A, or SIRT6.
[0246] In some embodiment, the functional domain may be a HDAC
Recruiter Effector Domain. Preferred examples include those in the
Table below, namely MeCP2, MBD2b, Sin3a, NcoR, SALL1, RCOR1. NcoR
is exemplified in the present Examples and, although preferred, it
is envisaged that others in the class will also be useful.
TABLE-US-00006 Table of HDAC Recruiter Effector Domains Complex
Full Selected Final Substrate Modification size truncation size
Catalytic Subtype Name (if known) (if known) Organism (aa) (aa)
(aa) domain Sin3a MeCP2 -- -- R. 492 207-492 286 -- norvegicus
(Nan) Sin3a MBD2b -- -- H. 262 45-262 218 -- sapiens (Boeke) Sin3a
SinSa -- -- H. 1273 524-851 328 627-829: HDAC1 sapiens (Laherty)
interaction NcoR NcoR -- -- H. 2440 420-488 69 -- sapiens (Zhang)
NuRD SALL1 -- -- M. 1322 1-93 93 -- musculus (Lauberth) CoREST
RCOR1 -- -- H. 482 81-300 220 -- sapiens (Gu, Ouyang)
[0247] In some embodiment, the functional domain may be a
Methyltransferase (HMT) Effector Domain. Preferred examples include
those in the Table below, namely NUE, vSET, EHMT2/G9A, SUV39H1,
dim-5, KYP, SUVR4, SET4, SET1, SETD8, and TgSET8. NUE is
exemplified in the present Examples and, although preferred, it is
envisaged that others in the class will also be useful.
TABLE-US-00007 Table of Histone Methyltransferase (HMT) Effector
Domains Complex Full Selected Final Substrate Modification size
truncation size Catalytic Subtype Name (if known) (if known)
Organism (aa) (aa) (aa) domain SET NUE H2B, -- C. 219 1-219 219 --
H3, H4 trachomatis (Pennini) SET vSET -- H3K27me3 P. 119 1-119 119
4-112: SET2 bursaria (Mujtaba) chlorella virus SUV39 EHMT2/G9A
H1.4K2, H3K9me1/2, M. 1263 969-1263 295 1025-1233: preSET, family
H3K9, H1K25me1 musculus (Tachibana) SET, postSET H3K27 SUV39
SUV39H1 -- H3K9me2/3 H. 412 79-412 334 172-412: preSET, sapiens
(Snowden) SET, postSET Suvar3-9 dim-5 -- H3K9me3 N. 331 1-331 331
77-331: preSET, crassa (Rathert) SET, postSET Suvar3-9 KYP --
H3K9me1/2 A. 624 335-601 267 -- (SUVH thaliana (Jackson) subfamily)
Suvar3-9 SUVR4 H3K9me1 H3K9me2/3 A. 492 180-492 313 192-462:
preSET, (SUVR thaliana (Thorstensen) SET, postSET subfamily)
Suvar4-20 SET4 -- H4K20me3 C. 288 1-288 288 -- elegans (Vielle)
SET8 SET1 -- H4K20me1 C. 242 1-242 242 -- elegans (Vielle) SET8
SETD8 -- H4K20me1 H. 393 185-393 209 256-382: SET sapiens (Couture)
SET8 TgSET8 -- H4K20me1/2/3 T. 1893 1590-1893 304 1749-1884: SET
gondii (Sautel)
[0248] In some embodiment, the functional domain may be a Histone
Methyltransferase (HMT) Recruiter Effector Domain. Preferred
examples include those in the Table below, namely Hp 1 a, PHF19,
and NIPP1.
TABLE-US-00008 Table of Histone Methyltransferase (HMT) Recruiter
Effector Domains Complex Full Selected Final Substrate Modification
size truncation size Catalytic Subtype Name (if known) (if known)
Organism (aa) (aa) (aa) domain -- Hp1a -- H3K9me3 M. 191 73-191 119
121-179: chromoshadow musculus (Hathaway) -- PHF19 -- H3K27me3 H.
580 (1-250) + GGSG 335 163-250: PHD2 sapiens linker (SEQ ID NO:
(Ballare) 43) + (500-580) -- NIPP1 -- H3K27me3 H. 351 1-329 329
310-329: EED sapiens (Jin)
[0249] In some embodiment, the functional domain may be Histone
Acetyltransferase Inhibitor Effector Domain. Preferred examples
include SET/TAF-1.beta. listed in the Table below.
TABLE-US-00009 Table of Histone Acetyltransferase Inhibitor
Effector Domains Complex Full Selected Final Substrate Modification
size truncation size Catalytic Subtype Name (if known) (if known)
Organism (aa) (aa) (aa) domain -- SET/TAF-1.beta. -- -- M. 289
1-289 289 -- musculus (Cervoni)
[0250] It is also preferred to target endogenous (regulatory)
control elements (such as enhancers and silencers) in addition to a
promoter or promoter-proximal elements. Thus, the invention can
also be used to target endogenous control elements (including
enhancers and silencers) in addition to targeting of the promoter.
These control elements can be located upstream and downstream of
the transcriptional start site (TSS), starting from 200 bp from the
TSS to 100 kb away. Targeting of known control elements can be used
to activate or repress the gene of interest. In some cases, a
single control element can influence the transcription of multiple
target genes. Targeting of a single control element could therefore
be used to control the transcription of multiple genes
simultaneously.
[0251] Targeting of putative control elements on the other hand
(e.g. by tiling the region of the putative control element as well
as 200 bp up to 100 kB around the element) can be used as a means
to verify such elements (by measuring the transcription of the gene
of interest) or to detect novel control elements (e.g. by tiling
100 kb upstream and downstream of the TSS of the gene of interest).
In addition, targeting of putative control elements can be useful
in the context of understanding genetic causes of disease. Many
mutations and common SNP variants associated with disease
phenotypes are located outside coding regions. Targeting of such
regions with either the activation or repression systems described
herein can be followed by readout of transcription of either a) a
set of putative targets (e.g. a set of genes located in closest
proximity to the control element) or b) whole-transcriptome readout
by e.g. RNAseq or microarray. This would allow for the
identification of likely candidate genes involved in the disease
phenotype. Such candidate genes could be useful as novel drug
targets.
[0252] Histone acetyltransferase (HAT) inhibitors are mentioned
herein. However, an alternative in some embodiments is for the one
or more functional domains to comprise an acetyltransferase,
preferably a histone acetyltransferase. These are useful in the
field of epigenomics, for example in methods of interrogating the
epigenome. Methods of interrogating the epigenome may include, for
example, targeting epigenomic sequences. Targeting epigenomic
sequences may include the guide being directed to an epigenomic
target sequence. Epigenomic target sequence may include, in some
embodiments, include a promoter, silencer or an enhancer
sequence.
[0253] Use of a functional domain linked to a CRISPR-Cas enzyme as
described herein, preferably a dead-Cas, more preferably a
dead-Cas9, to target epigenomic sequences can be used to activate
or repress promoters, silencer or enhancers.
[0254] Examples of acetyltransferases are known but may include, in
some embodiments, histone acetyltransferases. In some embodiments,
the histone acetyltransferase may comprise the catalytic core of
the human acetyltransferase p300 (Gerbasch & Reddy, Nature
Biotech 6 Apr. 2015).
[0255] In some preferred embodiments, the functional domain is
linked to a dead-Cas9 enzyme to target and activate epigenomic
sequences such as promoters or enhancers. One or more guides
directed to such promoters or enhancers may also be provided to
direct the binding of the CRISPR enzyme to such promoters or
enhancers.
[0256] The term "associated with" is used here in relation to the
association of the functional domain to the CRISPR enzyme or the
adaptor protein. It is used in respect of how one molecule
`associates` with respect to another, for example between an
adaptor protein and a functional domain, or between the CRISPR
enzyme and a functional domain. In the case of such protein-protein
interactions, this association may be viewed in terms of
recognition in the way an antibody recognizes an epitope.
Alternatively, one protein may be associated with another protein
via a fusion of the two, for instance one subunit being fused to
another subunit. Fusion typically occurs by addition of the amino
acid sequence of one to that of the other, for instance via
splicing together of the nucleotide sequences that encode each
protein or subunit. Alternatively, this may essentially be viewed
as binding between two molecules or direct linkage, such as a
fusion protein. In any event, the fusion protein may include a
linker between the two subunits of interest (i.e. between the
enzyme and the functional domain or between the adaptor protein and
the functional domain). Thus, in some embodiments, the CRISPR
enzyme or adaptor protein is associated with a functional domain by
binding thereto. In other embodiments, the CRISPR enzyme or adaptor
protein is associated with a functional domain because the two are
fused together, optionally via an intermediate linker.
[0257] Attachment of a functional domain or fusion protein can be
via a linker, e.g., a flexible glycine-serine (GlyGlyGlySer) (SEQ
ID NO: 28) or (GGGS)3 (SEQ ID NO: 29) or a rigid alpha-helical
linker such as (Ala(GluAlaAlaAlaLys)Ala) (SEQ ID NO: 30). Linkers
such as (GGGGS)3 (SEQ ID NO: 27) are preferably used herein to
separate protein or peptide domains. (GGGGS)3 (SEQ ID NO: 27) is
preferable because it is a relatively long linker (15 amino acids).
The glycine residues are the most flexible and the serine residues
enhance the chance that the linker is on the outside of the
protein. (GGGGS)6 (SEQ ID NO: 31) (GGGGS)9 (SEQ ID NO: 32) or
(GGGGS)12 (SEQ ID NO: 33) may preferably be used as alternatives.
Other preferred alternatives are (GGGGS)1 (SEQ ID NO: 34), (GGGGS)2
(SEQ ID NO: 35), (GGGGS)4 (SEQ ID NO: 36), (GGGGS)5 (SEQ ID NO:
37), (GGGGS)7 (SEQ ID NO: 38), (GGGGS)8 (SEQ ID NO: 39), (GGGGS)10
(SEQ ID NO: 40), or (GGGGS)11 (SEQ ID NO: 41). Alternative linkers
are available, but highly flexible linkers are thought to work best
to allow for maximum opportunity for the 2 parts of the Cas9 to
come together and thus reconstitute Cas9 activity. One alternative
is that the NLS of nucleoplasmin can be used as a linker. For
example, a linker can also be used between the Cas9 and any
functional domain. Again, a (GGGGS)3 linker (SEQ ID NO: 27) may be
used here (or the 6, 9, or 12 repeat versions therefore) or the NLS
of nucleoplasmin can be used as a linker between Cas9 and the
functional domain.
[0258] It is also preferred to target endogenous (regulatory)
control elements (such as enhancers and silencers) in addition to a
promoter or promoter-proximal elements. Thus, the invention can
also be used to target endogenous control elements (including
enhancers and silencers) in addition to targeting of the promoter.
These control elements can be located upstream and downstream of
the transcriptional start site (TSS), starting from 200 bp from the
TSS to 100 kb away. Targeting of known control elements can be used
to activate or repress the gene of interest. In some cases, a
single control element can influence the transcription of multiple
target genes. Targeting of a single control element could therefore
be used to control the transcription of multiple genes
simultaneously.
[0259] Targeting of putative control elements on the other hand
(e.g. by tiling the region of the putative control element as well
as 200 bp up to 100 kB around the element) can be used as a means
to verify such elements (by measuring the transcription of the gene
of interest) or to detect novel control elements (e.g. by tiling
100 kb upstream and downstream of the TSS of the gene of interest).
In addition, targeting of putative control elements can be useful
in the context of understanding genetic causes of disease. Many
mutations and common SNP variants associated with disease
phenotypes are located outside coding regions. Targeting of such
regions with either the activation or repression systems described
herein can be followed by readout of transcription of either a) a
set of putative targets (e.g. a set of genes located in closest
proximity to the control element) or b) whole-transcriptome readout
by e.g. RNAseq or microarray. This would allow for the
identification of likely candidate genes involved in the disease
phenotype. Such candidate genes could be useful as novel drug
targets.
[0260] The term "associated with" is used here in relation to the
association of the functional domain to the CRISPR enzyme or the
adaptor protein. It is used in respect of how one molecule
`associates` with respect to another, for example between an
adaptor protein and a functional domain, or between the CRISPR
enzyme and a functional domain. In the case of such protein-protein
interactions, this association may be viewed in terms of
recognition in the way an antibody recognizes an epitope.
Alternatively, one protein may be associated with another protein
via a fusion of the two, for instance one subunit being fused to
another subunit. Fusion typically occurs by addition of the amino
acid sequence of one to that of the other, for instance via
splicing together of the nucleotide sequences that encode each
protein or subunit. Alternatively, this may essentially be viewed
as binding between two molecules or direct linkage, such as a
fusion protein. In any event, the fusion protein may include a
linker between the two subunits of interest (i.e. between the
enzyme and the functional domain or between the adaptor protein and
the functional domain). Thus, in some embodiments, the CRISPR
enzyme or adaptor protein is associated with a functional domain by
binding thereto. In other embodiments, the CRISPR enzyme or adaptor
protein is associated with a functional domain because the two are
fused together, optionally via an intermediate linker.
[0261] In an embodiment, nucleic acid molecule(s) encoding the
DNA-targeting effector protein, in particular Cas9 or an ortholog
or homolog thereof, may be codon-optimized for expression in a
eukaryotic cell. A eukaryote can be as herein discussed. Nucleic
acid molecule(s) can be engineered or non-naturally occurring.
[0262] In an embodiment, the DNA-targeting effector protein, in
particular Cas9, may comprise one or more mutations (and hence
nucleic acid molecule(s) coding for same may have mutation(s). The
mutations may be artificially introduced mutations and may include
but are not limited to one or more mutations in a catalytic domain.
Examples of catalytic domains with reference to a Cas9 enzyme may
include but are not limited to RuvC I, RuvC II, RuvC III and HNH
domains.
[0263] In an embodiment, the Type II protein such as Cas9 may
comprise one or more mutations. The mutations may be artificially
introduced mutations and may include but are not limited to one or
more mutations in a catalytic domain, to provide a nickase, for
example. Examples of catalytic domains with reference to a Cas
enzyme may include but are not limited to RuvC I, RuvC II, RuvC
III, and HNH domains.
[0264] In an embodiment, the Type II protein such as Cas9 may be
used as a generic nucleic acid binding protein with fusion to or
being operably linked to a functional domain. Exemplary functional
domains may include but are not limited to translational initiator,
translational activator, translational repressor, nucleases, in
particular ribonucleases, a spliceosome, beads, a light
inducible/controllable domain or a chemically
inducible/controllable domain.
[0265] In some embodiments, the unmodified nucleic acid-targeting
effector protein may have cleavage activity. In some embodiments,
the DNA-targeting effector protein may direct cleavage of one or
both nucleic acid strands at the location of or near a target
sequence, such as within the target sequence and/or within the
complement of the target sequence or at sequences associated with
the target sequence. In some embodiments, the nucleic
acid-targeting Cas9 protein may direct cleavage of one or both DNA
or RNA strands within about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20,
25, 50, 100, 200, 500, or more base pairs from the first or last
nucleotide of a target sequence. In some embodiments, the cleavage
may be blunt, i.e., generating blunt ends. In some embodiments, the
cleavage may be staggered, i.e., generating sticky ends. In some
embodiments, the cleavage may be a staggered cut with a 5'
overhang, e.g., a 5' overhang of 1 to 5 nucleotides. In some
embodiments, the cleavage may be a staggered cut with a 3'
overhang, e.g., a 3' overhang of 1 to 5 nucleotides. In some
embodiments, a vector encodes a nucleic acid-targeting Cas9 protein
that may be mutated with respect to a corresponding wild-type
enzyme such that the mutated nucleic acid-targeting Cas9 protein
lacks the ability to cleave one or both DNA or RNA strands of a
target polynucleotide containing a target sequence. As a further
example, two or more catalytic domains of Cas9 (RuvC I, RuvC II,
and RuvC III or the HNH domain) may be mutated to produce a mutated
Cas substantially lacking all RNA cleavage activity. As described
herein, corresponding catalytic domains of a Cas9 effector protein
may also be mutated to produce a mutated Cas9 lacking all DNA
cleavage activity or having substantially reduced DNA cleavage
activity. In some embodiments, a nucleic acid-targeting effector
protein may be considered to substantially lack all RNA cleavage
activity when the RNA cleavage activity of the mutated enzyme is
about no more than 25%, 10%, 5%, 1%, 0.1%, 0.01%, or less of the
nucleic acid cleavage activity of the non-mutated form of the
enzyme; an example can be when the nucleic acid cleavage activity
of the mutated form is nil or negligible as compared with the
non-mutated form. An effector protein may be identified with
reference to the general class of enzymes that share homology to
the biggest nuclease with multiple nuclease domains from the Type
II CRISPR system. Most preferably, the effector protein is a Type
II protein such as Cas9. By derived, Applicants mean that the
derived enzyme is largely based, in the sense of having a high
degree of sequence homology with, a wildtype enzyme, but that it
has been mutated (modified) in some way as known in the art or as
described herein.
[0266] Again, it will be appreciated that the terms Cas9 and CRISPR
enzyme and CRISPR protein and Cas9 protein are generally used
interchangeably and at all points of reference herein refer by
analogy to novel CRISPR effector proteins further described in this
application, unless otherwise apparent. As mentioned above, many of
the residue numberings used herein refer to the effector protein
from the Type II CRISPR locus. However, it will be appreciated that
this invention includes many more effector proteins from other
species of microbes.
[0267] In certain embodiments, Cas9 may be constitutively present
or inducibly present or conditionally present or administered or
delivered. Cas9 optimization may be used to enhance function or to
develop new functions, one can generate chimeric Cas proteins. And
Cas may be used as a generic nucleic acid binding protein.
[0268] Typically, in the context of an endogenous nucleic
acid-targeting system, formation of a nucleic acid-targeting
complex (comprising a guide RNA hybridized to a target sequence and
complexed with one or more nucleic acid-targeting effector
proteins) results in cleavage of one or both DNA or RNA strands in
or near (e.g., within 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 50, or
more base pairs from) the target sequence. As used herein the term
"sequence(s) associated with a target locus of interest" refers to
sequences near the vicinity of the target sequence (e.g. within 1,
2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 50, or more base pairs from the
target sequence, wherein the target sequence is comprised within a
target locus of interest).
[0269] An example of a codon optimized sequence, is in this
instance a sequence optimized for expression in a eukaryote, e.g.,
humans (i.e. being optimized for expression in humans), or for
another eukaryote, animal or mammal as herein discussed; see, e.g.,
SaCas9 human codon optimized sequence in WO 2014/093622
(PCT/US2013/074667) as an example of a codon optimized sequence
(from knowledge in the art and this disclosure, codon optimizing
coding nucleic acid molecule(s), especially as to effector protein
(e.g., Cas9) is within the ambit of the skilled artisan). Whilst
this is preferred, it will be appreciated that other examples are
possible and codon optimization for a host species other than
human, or for codon optimization for specific organs is known. In
some embodiments, an enzyme coding sequence encoding a
DNA-targeting Cas protein is codon optimized for expression in
particular cells, such as eukaryotic cells. The eukaryotic cells
may be those of or derived from a particular organism, such as a
mammal, including but not limited to human, or non-human eukaryote
or animal or mammal as herein discussed, e.g., mouse, rat, rabbit,
dog, livestock, or non-human mammal or primate. In some
embodiments, processes for modifying the germ line genetic identity
of human beings and/or processes for modifying the genetic identity
of animals which are likely to cause them suffering without any
substantial medical benefit to man or animal, and also animals
resulting from such processes, may be excluded. In general, codon
optimization refers to a process of modifying a nucleic acid
sequence for enhanced expression in the host cells of interest by
replacing at least one codon (e.g., about or more than about 1, 2,
3, 4, 5, 10, 15, 20, 25, 50, or more codons) of the native sequence
with codons that are more frequently or most frequently used in the
genes of that host cell while maintaining the native amino acid
sequence. Various species exhibit particular bias for certain
codons of a particular amino acid. Codon bias (differences in codon
usage between organisms) often correlates with the efficiency of
translation of messenger RNA (mRNA), which is in turn believed to
be dependent on, among other things, the properties of the codons
being translated and the availability of particular transfer RNA
(tRNA) molecules. The predominance of selected tRNAs in a cell is
generally a reflection of the codons used most frequently in
peptide synthesis. Accordingly, genes can be tailored for optimal
gene expression in a given organism based on codon optimization.
Codon usage tables are readily available, for example, at the
"Codon Usage Database" available at www.kazusa.orjp/codon/ and
these tables can be adapted in a number of ways. See Nakamura, Y.,
et al. "Codon usage tabulated from the international DNA sequence
databases: status for the year 2000" Nucl. Acids Res. 28:292
(2000). Computer algorithms for codon optimizing a particular
sequence for expression in a particular host cell are also
available, such as Gene Forge (Aptagen; Jacobus, Pa.), are also
available. In some embodiments, one or more codons (e.g., 1, 2, 3,
4, 5, 10, 15, 20, 25, 50, or more, or all codons) in a sequence
encoding a DNA/RNA-targeting Cas9 protein corresponds to the most
frequently used codon for a particular amino acid.
[0270] In one aspect, the invention provides methods for using one
or more elements of a nucleic acid-targeting system. The nucleic
acid-targeting complex of the invention provides an effective means
for modifying a target DNA (single or double stranded, linear or
super-coiled). The nucleic acid-targeting complex of the invention
has a wide variety of utility including modifying (e.g., deleting,
inserting, translocating, inactivating, activating) a target DNA in
a multiplicity of cell types. As such the nucleic acid-targeting
complex of the invention has a broad spectrum of applications in,
e.g., gene therapy, drug screening, disease diagnosis, and
prognosis. An exemplary nucleic acid-targeting complex comprises a
DNA-targeting effector protein complexed with a guide RNA
hybridized to a target sequence within the target locus of
interest.
[0271] In one embodiment, this invention provides a method of
cleaving a target DNA that encodes an RNA. The method may comprise
modifying a target DNA using a nucleic acid-targeting complex that
binds to the target DNA and effect cleavage of said target DNA. The
encoded RNA can be any RNA endogenous or exogenous to the
eukaryotic cell. For example, the target RNA can be a RNA residing
in the nucleus of the eukaryotic cell. The target RNA can be a
sequence (e.g., mRNA or pre-mRNA) coding a gene product (e.g., a
protein) or a non-coding sequence (e.g., ncRNA, lncRNA, tRNA, or
rRNA). Examples of encoded RNA include a sequence associated with a
signaling biochemical pathway, e.g., a signaling biochemical
pathway-associated RNA. Examples of encoded RNA include a disease
associated RNA. A "disease-associated" RNA refers to any RNA which
is yielding translation products at an abnormal level or in an
abnormal form in cells derived from a disease-affected tissues
compared with tissues or cells of a non disease control. It may be
a RNA transcribed from a gene that becomes expressed at an
abnormally high level; it may be a RNA transcribed from a gene that
becomes expressed at an abnormally low level, where the altered
expression correlates with the occurrence and/or progression of the
disease. A disease-associated RNA also refers to a RNA transcribed
from a gene possessing mutation(s) or genetic variation that is
directly responsible or is in linkage disequilibrium with a gene(s)
that is responsible for the etiology of a disease. The translated
products may be known or unknown, and may be at a normal or
abnormal level. The encoded RNA of a DNA-targeting complex can be
any RNA endogenous or exogenous to the eukaryotic cell. For
example, the target RNA can be a RNA residing in the nucleus of the
eukaryotic cell. The encoded RNA can be a sequence (e.g., mRNA or
pre-mRNA) coding a gene product (e.g., a protein) or a non-coding
sequence (e.g., ncRNA, lncRNA, tRNA, or rRNA).
[0272] In some embodiments, the method may comprise allowing a
nucleic acid-targeting complex to bind to the target DNA to effect
cleavage of said target DNA thereby modifying the target DNA,
wherein the nucleic acid-targeting complex comprises a nucleic
acid-targeting effector protein complexed with a guide RNA
hybridized to a target sequence within said target DNA. In one
aspect, the invention provides a method of modifying expression of
DNA in a eukaryotic cell. In some embodiments, the method comprises
allowing a nucleic acid-targeting complex to bind to the DNA such
that said binding results in increased or decreased expression of
said DNA; wherein the nucleic acid-targeting complex comprises a
nucleic acid-targeting effector protein complexed with a guide RNA.
Similar considerations and conditions apply as above for methods of
modifying a target DNA. In fact, these sampling, culturing and
re-introduction options apply across the aspects of the present
invention. In one aspect, the invention provides for methods of
modifying a target DNA in a eukaryotic cell, which may be in vivo,
ex vivo or in vitro. In some embodiments, the method comprises
sampling a cell or population of cells from a human or non-human
animal, and modifying the cell or cells. Culturing may occur at any
stage ex vivo. The cell or cells may even be re-introduced into the
non-human animal or plant. For re-introduced cells it is
particularly preferred that the cells are stem cells.
[0273] Indeed, in any aspect of the invention, the nucleic
acid-targeting complex may comprise a nucleic acid-targeting
effector protein complexed with a guide RNA hybridized to a target
sequence.
[0274] The invention relates to the engineering and optimization of
systems, methods and compositions used for the control of gene
expression involving DNA sequence targeting, that relate to the
nucleic acid-targeting system and components thereof. An advantage
of the present methods is that the CRISPR system minimizes or
avoids off-target binding and its resulting side effects. This is
achieved using systems arranged to have a high degree of sequence
specificity for the target DNA. In relation to a nucleic
acid-targeting complex or system preferably, the tracr sequence has
one or more hairpins and is 30 or more nucleotides in length, 40 or
more nucleotides in length, or 50 or more nucleotides in length;
the crRNA sequence is between 10 to 30 nucleotides in length, the
nucleic acid-targeting effector protein is a Type II Cas9 effector
protein.
Crystallization of CRISPR-Cas9 and Characterization of Crystal
Structure
[0275] The crystals of the Cas9 can be obtained by techniques of
protein crystallography, including batch, liquid bridge, dialysis,
vapor diffusion and hanging drop methods. Generally, the crystals
of the invention are grown by dissolving substantially pure
CRISPR-Cas9 and a nucleic acid molecule to which it binds in an
aqueous buffer containing a precipitant at a concentration just
below that necessary to precipitate. Water is removed by controlled
evaporation to produce precipitating conditions, which are
maintained until crystal growth ceases. The crystal structure
information is described in U.S. provisional applications
61/915,251 filed Dec. 12, 2013, 61/930,214 filed on Jan. 22, 2014,
61/980,012 filed Apr. 15, 2014 and international application
PCT/US2014/069925, filed Dec. 12, 2014; and Nishimasu et al,
"Crystal Structure of Cas9 in Complex with Guide RNA and Target
DNA," Cell 156(5):935-949, DOI:
http://dx.doi.org/10.1016/j.cell.2014.02.001 (2014), each and all
of which are incorporated herein by reference.
[0276] Uses of the Crystals, Crystal Structure and Atomic Structure
Co-Ordinates: The crystals of the Cas9, and particularly the atomic
structure co-ordinates obtained therefrom, have a wide variety of
uses. The crystals and structure co-ordinates are particularly
useful for identifying compounds (nucleic acid molecules) that bind
to CRISPR-Cas9, and CRISPR-Cas9s that can bind to particular
compounds (nucleic acid molecules). Thus, the structure
co-ordinates described herein can be used as phasing models in
determining the crystal structures of additional synthetic or
mutated CRISPR-Cas9s, Cas9s, nickases, binding domains. The
provision of the crystal structure of CRISPR-Cas9 complexed with a
nucleic acid molecule as applied in conjunction with the herein
teachings provides the skilled artisan with a detailed insight into
the mechanisms of action of CRISPR-Cas9. This insight provides a
means to design modified CRISPR-Cas9s, such as by attaching thereto
a functional group, such as a repressor or activator. While one can
attach a functional group such as a repressor or activator to the N
or C terminal of CRISPR-Cas9, the crystal structure demonstrates
that the N terminal seems obscured or hidden, whereas the C
terminal is more available for a functional group such as repressor
or activator. Moreover, the crystal structure demonstrates that
there is a flexible loop between approximately CRISPR-Cas9 (S.
pyogenes) residues 534-676 which is suitable for attachment of a
functional group such as an activator or repressor. Attachment can
be via a linker, e.g., a flexible glycine-serine (GlyGlyGlySer)
(SEQ ID NO: 28) or (GGGS).sub.3 (SEQ ID NO: 29) or a rigid
alpha-helical linker such as (Ala(GluAlaAlaAlaLys)Ala) (SEQ ID NO:
30). In addition to the flexible loop there is also a nuclease or
H3 region, an H2 region and a helical region. By "helix" or
"helical", is meant a helix as known in the art, including, but not
limited to an alpha-helix. Additionally, the term helix or helical
may also be used to indicate a c-terminal helical element with an
N-terminal turn.
[0277] The provision of the crystal structure of CRISPR-Cas9
complexed with a nucleic acid molecule allows a novel approach for
drug or compound discovery, identification, and design for
compounds that can bind to CRISPR-Cas9 and thus the invention
provides tools useful in diagnosis, treatment, or prevention of
conditions or diseases of multicellular organisms, e.g., algae,
plants, invertebrates, fish, amphibians, reptiles, avians, mammals;
for example domesticated plants, animals (e.g., production animals
such as swine, bovine, chicken; companion animal such as felines,
canines, rodents (rabbit, gerbil, hamster); laboratory animals such
as mouse, rat), and humans.
[0278] In any event, the determination of the three-dimensional
structure of CRISPR-Cas 9 (S. pyogenes Cas9) complex provides a
basis for the design of new and specific nucleic acid molecules
that bind to CRISPR-Cas 9 (e.g., S. pyogenes Cas9), as well as the
design of new CRISPR-Cas9 systems, such as by way of modification
of the CRISPR-Cas9 system to bind to various nucleic acid
molecules, by way of modification of the CRISPR-Cas9 system to have
linked thereto to any one or more of various functional groups that
may interact with each other, with the CRISPR-Cas9 (e.g., an
inducible system that provides for self-activation and/or
self-termination of function), with the nucleic acid molecule
nucleic acid molecules (e.g., the functional group may be a
regulatory or functional domain which may be selected from the
group consisting of a transcriptional repressor, a transcriptional
activator, a nuclease domain, a DNA methyl transferase, a protein
acetyltransferase, a protein deacetylase, a protein
methyltransferase, a protein deaminase, a protein kinase, and a
protein phosphatase; and, in some aspects, the functional domain is
an epigenetic regulator; see, e.g., Zhang et al., U.S. Pat. No.
8,507,272, and it is again mentioned that it and all documents
cited herein and all appln cited documents are hereby incorporated
herein by reference), by way of modification of Cas9, by way of
novel nickases). Indeed, the herewith CRISPR-Cas9 (S. pyogenes
Cas9) crystal structure has a multitude of uses. For example, from
knowing the three-dimensional structure of CRISPR-Cas9 (S. pyogenes
Cas9) crystal structure, computer modelling programs may be used to
design or identify different molecules expected to interact with
possible or confirmed sites such as binding sites or other
structural or functional features of the CRISPR-Cas9 system (e.g.,
S. pyogenes Cas9). Compound that potentially bind ("binder") can be
examined through the use of computer modeling using a docking
program. Docking programs are known; for example GRAM, DOCK or
AUTODOCK (see Walters et al. Drug Discovery Today, vol. 3, no. 4
(1998), 160-178, and Dunbrack et al. Folding and Design 2 (1997),
27-42). This procedure can include computer fitting of potential
binders ascertain how well the shape and the chemical structure of
the potential binder will bind to a CRISPR-Cas9 system (e.g., S.
pyogenes Cas9). Computer-assisted, manual examination of the active
site or binding site of a CRISPR-Cas9 system (e.g., S. pyogenes
Cas9) may be performed. Programs such as GRID (P. Goodford, J. Med.
Chem, 1985, 28, 849-57)--a program that determines probable
interaction sites between molecules with various functional
groups--may also be used to analyze the active site or binding site
to predict partial structures of binding compounds. Computer
programs can be employed to estimate the attraction, repulsion or
steric hindrance of the two binding partners, e.g., CRISPR-Cas9
system (e.g., S. pyogenes Cas9) and a candidate nucleic acid
molecule or a nucleic acid molecule and a candidate CRISPR-Cas9
system (e.g., S. pyogenes Cas9); and the CRISPR-Cas9 crystal
structure (S. pyogenes Cas9) herewith enables such methods.
Generally, the tighter the fit, the fewer the steric hindrances,
and the greater the attractive forces, the more potent the
potential binder, since these properties are consistent with a
tighter binding constant. Furthermore, the more specificity in the
design of a candidate CRISPR-Cas9 system (e.g., S. pyogenes Cas9),
the more likely it is that it will not interact with off-target
molecules as well. Also, "wet" methods are enabled by the instant
invention. For example, in an aspect, the invention provides for a
method for determining the structure of a binder (e.g., target
nucleic acid molecule) of a candidate CRISPR-Cas9 system (e.g., S.
pyogenes Cas9) bound to the candidate CRISPR-Cas9 system (e.g., S.
pyogenes Cas9), said method comprising, (a) providing a first
crystal of a candidate CRISPR-Cas9 system (S. pyogenes Cas9)
according to the invention or a second crystal of a candidate a
candidate CRISPR-Cas9 system (e.g., S. pyogenes Cas9), (b)
contacting the first crystal or second crystal with said binder
under conditions whereby a complex may form; and (c) determining
the structure of said a candidate (e.g., CRISPR-Cas9 system (e.g.,
S. pyogenes Cas9) or CRISPR-Cas9 system (S. pyogenes Cas9) complex.
The second crystal may have essentially the same coordinates
discussed herein, however due to minor alterations in CRISPR-Cas9
system (e.g., from the Cas9 of such a system being e.g., S.
pyogenes Cas9 versus being S. pyogenes Cas9), wherein "e.g., S.
pyogenes Cas9" indicates that the Cas9 is a Cas9 and can be of or
derived from S. pyogenes or an ortholog thereof), the crystal may
form in a different space group.
[0279] The invention further involves, in place of or in addition
to "in silico" methods, other "wet" methods, including high
throughput screening of a binder (e.g., target nucleic acid
molecule) and a candidate CRISPR-Cas9 system (e.g., S. pyogenes
Cas9), or a candidate binder (e.g., target nucleic acid molecule)
and a CRISPR-Cas9 system (e.g., S. pyogenes Cas9), or a candidate
binder (e.g., target nucleic acid molecule) and a candidate
CRISPR-Cas9 system (e.g., S. pyogenes Cas9) (the foregoing
CRISPR-Cas9 system(s) with or without one or more functional
group(s)), to select compounds with binding activity. Those pairs
of binder and CRISPR-Cas9 system which show binding activity may be
selected and further crystallized with the CRISPR-Cas9 crystal
having a structure herein, e.g., by co-crystallization or by
soaking, for X-ray analysis. The resulting X-ray structure may be
compared with that of the Cas9 Crystal Structure for a variety of
purposes, e.g., for areas of overlap. Having designed, identified,
or selected possible pairs of binder and CRISPR-Cas9 system by
determining those which have favorable fitting properties, e.g.,
predicted strong attraction based on the pairs of binder and
CRISPR-Cas9 crystal structure data herein, these possible pairs can
then be screened by "wet" methods for activity. Consequently, in an
aspect the invention can involve: obtaining or synthesizing the
possible pairs; and contacting a binder (e.g., target nucleic acid
molecule) and a candidate CRISPR-Cas9 system (e.g., S. pyogenes
Cas9), or a candidate binder (e.g., target nucleic acid molecule)
and a CRISPR-Cas9 system (e.g., S. pyogenes Cas9), or a candidate
binder (e.g., target nucleic acid molecule) and a candidate
CRISPR-Cas9 system (e.g., S. pyogenes Cas9) (the foregoing
CRISPR-Cas9 system(s) with or without one or more functional
group(s)) to determine ability to bind. In the latter step, the
contacting is advantageously under conditions to determine
function. Instead of, or in addition to, performing such an assay,
the invention may comprise: obtaining or synthesizing complex(es)
from said contacting and analyzing the complex(es), e.g., by X-ray
diffraction or NMR or other means, to determine the ability to bind
or interact. Detailed structural information can then be obtained
about the binding, and in light of this information, adjustments
can be made to the structure or functionality of a candidate
CRISPR-Cas9 system or components thereof. These steps may be
repeated and re-repeated as necessary. Alternatively or
additionally, potential CRISPR-Cas9 systems from or in the
foregoing methods can be with nucleic acid molecules in vivo,
including without limitation by way of administration to an
organism (including non-human animal and human) to ascertain or
confirm function, including whether a desired outcome (e.g.,
reduction of symptoms, treatment) results therefrom.
[0280] The invention further involves a method of determining three
dimensional structures of CRISPR-Cas systems or complex(es) of
unknown structure by using the structural co-ordinates of the Cas9
Crystal Structure. For example, if X-ray crystallographic or NMR
spectroscopic data are provided for a CRISPR-Cas system or complex
of unknown crystal structure, the structure of a CRISPR-Cas9
complex may be used to interpret that data to provide a likely
structure for the unknown system or complex by such techniques as
by phase modeling in the case of X-ray crystallography. Thus, an
inventive method can comprise: aligning a representation of the
CRISPR-Cas system or complex having an unknown crystal structure
with an analogous representation of the CRISPR-Cas(9) system and
complex of the crystal structure herein to match homologous or
analogous regions (e.g., homologous or analogous sequences);
modeling the structure of the matched homologous or analogous
regions (e.g., sequences) of the CRISPR-Cas system or complex of
unknown crystal structure based on the structure of the Cas9
Crystal Structure of the corresponding regions (e.g., sequences);
and, determining a conformation (e.g. taking into consideration
favorable interactions should be formed so that a low energy
conformation is formed) for the unknown crystal structure which
substantially preserves the structure of said matched homologous
regions. "Homologous regions" describes, for example as to amino
acids, amino acid residues in two sequences that are identical or
have similar, e.g., aliphatic, aromatic, polar, negatively charged,
or positively charged, side-chain chemical groups. Homologous
regions as to nucleic acid molecules can include at least 85% or
86% or 87% or 88% or 89% or 90% or 91% or 92% or 93% or 94% or 95%
or 96% or 97% or 98% or 99% homology or identity. Identical and
similar regions are sometimes described as being respectively
"invariant" and "conserved" by those skilled in the art. Homology
modeling is a technique that is well known to those skilled in the
art (see, e.g., Greer, Science vol. 228 (1985) 1055, and Blundell
et al. Eur J Biochem vol 172 (1988), 513). The computer
representation of the conserved regions of the CRISPR-Cas9 crystal
structure and those of a CRISPR-Cas system of unknown crystal
structure aid in the prediction and determination of the crystal
structure of the CRISPR-Cas system of unknown crystal
structure.
[0281] Further still, the aspects of the invention which employ the
CRISPR-Cas9 crystal structure in silico may be equally applied to
new CRISPR-Cas crystal structures divined by using the
herein-referenced CRISPR-Cas9 crystal structure. In this fashion, a
library of CRISPR-Cas crystal structures can be obtained. Rational
CRISPR-Cas system design is thus provided by the instant invention.
For instance, having determined a conformation or crystal structure
of a CRISPR-Cas system or complex, by the methods described herein,
such a conformation may be used in a computer-based methods herein
for determining the conformation or crystal structure of other
CRISPR-Cas systems or complexes whose crystal structures are yet
unknown. Data from all of these crystal structures can be in a
database, and the herein methods can be more robust by having
herein comparisons involving the herein crystal structure or
portions thereof be with respect to one or more crystal structures
in the library. The invention further provides systems, such as
computer systems, intended to generate structures and/or perform
rational design of a CRISPR-Cas system or complex. The system can
contain: atomic co-ordinate data according to the herein-referenced
Crystal Structure or be derived therefrom e.g., by modeling, said
data defining the three-dimensional structure of a CRISPR-Cas
system or complex or at least one domain or sub-domain thereof, or
structure factor data therefor, said structure factor data being
derivable from the atomic co-ordinate data of the herein-referenced
Crystal Structure. The invention also involves computer readable
media with: atomic co-ordinate data according to the
herein-referenced Crystal Structure or derived therefrom e.g., by
homology modeling, said data defining the three-dimensional
structure of a CRISPR-Cas system or complex or at least one domain
or sub-domain thereof, or structure factor data therefor, said
structure factor data being derivable from the atomic co-ordinate
data of the herein-referenced Crystal Structure. "Computer readable
media" refers to any media which can be read and accessed directly
by a computer, and includes, but is not limited to: magnetic
storage media; optical storage media; electrical storage media;
cloud storage and hybrids of these categories. By providing such
computer readable media, the atomic co-ordinate data can be
routinely accessed for modeling or other "in silico" methods. The
invention further comprehends methods of doing business by
providing access to such computer readable media, for instance on a
subscription basis, via the Internet or a global
communication/computer network; or, the computer system can be
available to a user, on a subscription basis. A "computer system"
refers to the hardware means, software means and data storage means
used to analyze the atomic co-ordinate data of the present
invention. The minimum hardware means of computer-based systems of
the invention may comprise a central processing unit (CPU), input
means, output means, and data storage means. Desirably, a display
or monitor is provided to visualize structure data. The invention
further comprehends methods of transmitting information obtained in
any method or step thereof described herein or any information
described herein, e.g., via telecommunications, telephone, mass
communications, mass media, presentations, internet, email, etc.
The crystal structures of the invention can be analyzed to generate
Fourier electron density map(s) of CRISPR-Cas systems or complexes;
advantageously, the three-dimensional structure being as defined by
the atomic co-ordinate data according to the herein-referenced
Crystal Structure. Fourier electron density maps can be calculated
based on X-ray diffraction patterns. These maps can then be used to
determine aspects of binding or other interactions. Electron
density maps can be calculated using known programs such as those
from the CCP4 computer package (Collaborative Computing Project,
No. 4. The CCP4 Suite: Programs for Protein Crystallography, Acta
Crystallographica, D50, 1994, 760-763). For map visualization and
model building programs such as "QUANTA" (1994, San Diego, Calif.:
Molecular Simulations, Jones et al., Acta Crystallography A47
(1991), 110-119) can be used.
[0282] The herein-referenced Crystal Structure gives atomic
co-ordinate data for a CRISPR-Cas9 (S. pyogenes), and lists each
atom by a unique number; the chemical element and its position for
each amino acid residue (as determined by electron density maps and
antibody sequence comparisons), the amino acid residue in which the
element is located, the chain identifier, the number of the
residue, co-ordinates (e.g., X, Y, Z) which define with respect to
the crystallographic axes the atomic position (in angstroms) of the
respective atom, the occupancy of the atom in the respective
position, "B", isotropic displacement parameter (in
angstroms.sup.2) which accounts for movement of the atom around its
atomic center, and atomic number.
[0283] In particular embodiments of the invention, the
conformational variations in the crystal structures of the
CRISPR-Cas9 system or of components of the CRISPR-Cas9 provide
important and critical information about the flexibility or
movement of protein structure regions relative to nucleotide (RNA
or DNA) structure regions that may be important for CRISPR-Cas
system function. The structural information provided for Cas9 (e.g.
S. pyogenes Cas9) as the CRISPR enzyme in the present application
may be used to further engineer and optimize the CRISPR-Cas system
and this may be extrapolated to interrogate structure-function
relationships in other CRISPR enzyme systems as well. An aspect of
the invention relates to the crystal structure of S. pyogenes Cas9
in complex with sgRNA and its target DNA at 2.4 .ANG. resolution.
The structure revealed a bilobed architecture composed of target
recognition and nuclease lobes, accommodating a sgRNA:DNA duplex in
a positively-charged groove at their interface. The recognition
lobe is essential for sgRNA and DNA binding and the nuclease lobe
contains the HNH and RuvC nuclease domains, which are properly
positioned for the cleavage of complementary and non-complementary
strands of the target DNA, respectively. This high-resolution
structure and the functional analyses provided herein elucidate the
molecular mechanism of RNA-guided DNA targeting by Cas9, and
provides an abundance of information for generating optimized
CRISPR-Cas systems and components thereof
[0284] In particular embodiments of the invention, the crystal
structure provides a critical step towards understanding the
molecular mechanism of RNA-guided DNA targeting by Cas9. The
structural and functional analyses herein provide a useful scaffold
for rational engineering of Cas9-based genome modulating
technologies and may provide guidance as to Cas9-mediated
recognition of PAM sequences on the target DNA or mismatch
tolerance between the sgRNA:DNA duplex. Aspects of the invention
also relate to truncation mutants, e.g. an S. pyogenes Cas9
truncation mutant may facilitate packaging of Cas9 into
size-constrained viral vectors for in vivo and therapeutic
applications. Similarly, future engineering of the PAM Interacting
(PI) domain may allow programming of PAM specificity, improve
target site recognition fidelity, and increase the versatility of
the Cas9 genome engineering platform.
[0285] Accordingly, while the herein-referenced crystal structure
may be used in conjunction with the herein disclosure, and in
conjunction with the herein invention, the herein invention of
protected guides and the utility thereof could not have been
predicted from the herein-referenced crystal structure.
[0286] The invention comprehends optimized functional CRISPR-Cas
enzyme systems. In particular the CRISPR enzyme comprises one or
more mutations that converts it to a DNA binding protein to which
functional domains exhibiting a function of interest may be
recruited or appended or inserted or attached. In certain
embodiments, the CRISPR enzyme comprises one or more mutations
which include but are not limited to D10A, E762A, H840A, N854A,
N863A or D986A (based on the amino acid position numbering of a S.
pyogenes Cas9) and/or the one or more mutations is in a RuvC1 or
HNH domain of the CRISPR enzyme or is a mutation as otherwise as
discussed herein. In some embodiments, the CRISPR enzyme has one or
more mutations in a catalytic domain, wherein when transcribed, the
tracr mate sequence hybridizes to the tracr sequence and the guide
sequence directs sequence-specific binding of a CRISPR complex to
the target sequence, and wherein the enzyme further comprises a
functional domain.
[0287] The structural information provided herein allows for
interrogation of sgRNA (or chimeric RNA) interaction with the
target DNA and the CRISPR enzyme (e.g. Cas9) permitting engineering
or alteration of sgRNA structure to optimize functionality of the
entire CRISPR-Cas system. For example, loops of the sgRNA may be
extended, without colliding with the Cas9 protein by the insertion
of distinct RNA loop(s) or distinct sequence(s) that may recruit
adaptor proteins that can bind to the distinct RNA loop(s) or
distinct sequence(s). The adaptor proteins may include but are not
limited to orthogonal RNA-binding protein/aptamer combinations that
exist within the diversity of bacteriophage coat proteins. A list
of such coat proteins includes, but is not limited to: Q.beta., F2,
GA, fr, JP501, M12, R17, BZ13, JP34, JP500, KU1, M11, MX1, TW18,
VK, SP, FI, ID2, NL95, TW19, AP205, .phi.Cb5, .phi.Cb8r,
.phi.Cb12r, .phi.Cb23r, 7s and PRR1. These adaptor proteins or
orthogonal RNA binding proteins can further recruit effector
proteins or fusions which comprise one or more functional domains.
In some embodiments, the functional domain may be selected from the
group consisting of: transposase domain, integrase domain,
recombinase domain, resolvase domain, invertase domain, protease
domain, DNA methyltransferase domain, DNA hydroxylmethylase domain,
DNA demethylase domain, histone acetylase domain, histone
deacetylases domain, nuclease domain, repressor domain, activator
domain, nuclear-localization signal domains,
transcription-regulatory protein (or transcription complex
recruiting) domain, cellular uptake activity associated domain,
nucleic acid binding domain, antibody presentation domain, histone
modifying enzymes, recruiter of histone modifying enzymes;
inhibitor of histone modifying enzymes, histone methyltransferase,
histone demethylase, histone kinase, histone phosphatase, histone
ribosylase, histone deribosylase, histone ubiquitinase, histone
deubiquitinase, histone biotinase and histone tail protease.
[0288] In some preferred embodiments, the functional domain is a
transcriptional activation domain, preferably VP64. In some
embodiments, the functional domain is a transcription repression
domain, preferably KRAB. In some embodiments, the transcription
repression domain is SID, or concatemers of SID (eg SID4X). In some
embodiments, the functional domain is an epigenetic modifying
domain, such that an epigenetic modifying enzyme is provided. In
some embodiments, the functional domain is an activation domain,
which may be the P65 activation domain.
[0289] In one aspect surveyor analysis is used for identification
of indel activity/nuclease activity. In general survey analysis
includes extraction of genomic DNA, PCR amplification of the
genomic region flanking the CRISPR target site, purification of
products, re-annealing to enable heteroduplex formation. After
re-annealing, products are treated with SURVEYOR nuclease and
SURVEYOR enhancer S (Transgenomics) following the manufacturer's
recommended protocol. Analysis may be performed with
poly-acrylamide gels according to known methods. Quantification may
be based on relative band intensities.
Delivery Generally
[0290] Gene Editing or Altering a Target Loci with Cas9
[0291] The double strand break or single strand break in one of the
strands advantageously should be sufficiently close to target
position such that correction occurs. In an embodiment, the
distance is not more than 50, 100, 200, 300, 350 or 400
nucleotides. While not wishing to be bound by theory, it is
believed that the break should be sufficiently close to target
position such that the break is within the region that is subject
to exonuclease-mediated removal during end resection. If the
distance between the target position and a break is too great, the
mutation may not be included in the end resection and, therefore,
may not be corrected, as the template nucleic acid sequence may
only be used to correct sequence within the end resection
region.
[0292] In an embodiment, in which a guide RNA and a Type II
molecule, in particular Cas9 or an ortholog or homolog thereof,
preferably a Cas9 nuclease induce a double strand break for the
purpose of inducing HDR-mediated correction, the cleavage site is
between 0-200 bp (e.g., 0 to 175, 0 to 150, 0 to 125, 0 to 100, 0
to 75, 0 to 50, 0 to 25, 25 to 200, 25 to 175, 25 to 150, 25 to
125, 25 to 100, 25 to 75, 25 to 50, 50 to 200, 50 to 175, 50 to
150, 50 to 125, 50 to 100, 50 to 75, 75 to 200, 75 to 175, 75 to
150, 75 to 125, 75 to 100 bp) away from the target position. In an
embodiment, the cleavage site is between 0-100 bp (e.g., 0 to 75, 0
to 50, 0 to 25, 25 to 100, 25 to 75, 25 to 50, 50 to 100, 50 to 75
or 75 to 100 bp) away from the target position. In a further
embodiment, two or more guide RNAs complexing with Cas9 or an
ortholog or homolog thereof, may be used to induce multiplexed
breaks for purpose of inducing HDR-mediated correction.
[0293] The homology arm should extend at least as far as the region
in which end resection may occur, e.g., in order to allow the
resected single stranded overhang to find a complementary region
within the donor template. The overall length could be limited by
parameters such as plasmid size or viral packaging limits. In an
embodiment, a homology arm may not extend into repeated elements.
Exemplary homology arm lengths include a least 50, 100, 250, 500,
750 or 1000 nucleotides.
[0294] Target position, as used herein, refers to a site on a
target nucleic acid or target gene (e.g., the chromosome) that is
modified by a Type II, in particular Cas9 or an ortholog or homolog
thereof, preferably Cas9 molecule-dependent process. For example,
the target position can be a modified Cas9 molecule cleavage of the
target nucleic acid and template nucleic acid directed
modification, e.g., correction, of the target position. In an
embodiment, a target position can be a site between two
nucleotides, e.g., adjacent nucleotides, on the target nucleic acid
into which one or more nucleotides is added. The target position
may comprise one or more nucleotides that are altered, e.g.,
corrected, by a template nucleic acid. In an embodiment, the target
position is within a target sequence (e.g., the sequence to which
the guide RNA binds). In an embodiment, a target position is
upstream or downstream of a target sequence (e.g., the sequence to
which the guide RNA binds).
[0295] A template nucleic acid, as that term is used herein, refers
to a nucleic acid sequence which can be used in conjunction with a
Type II molecule, in particular Cas9 or an ortholog or homolog
thereof, preferably a Cas9 molecule and a guide RNA molecule to
alter the structure of a target position. In an embodiment, the
target nucleic acid is modified to have some or all of the sequence
of the template nucleic acid, typically at or near cleavage
site(s). In an embodiment, the template nucleic acid is single
stranded. In an alternate embodiment, the template nucleic acid is
double stranded. In an embodiment, the template nucleic acid is
DNA, e.g., double stranded DNA. In an alternate embodiment, the
template nucleic acid is single stranded DNA.
[0296] In an embodiment, the template nucleic acid alters the
structure of the target position by participating in homologous
recombination. In an embodiment, the template nucleic acid alters
the sequence of the target position. In an embodiment, the template
nucleic acid results in the incorporation of a modified, or
non-naturally occurring base into the target nucleic acid.
[0297] The template sequence may undergo a breakage mediated or
catalyzed recombination with the target sequence. In an embodiment,
the template nucleic acid may include sequence that corresponds to
a site on the target sequence that is cleaved by a Cas9 mediated
cleavage event. In an embodiment, the template nucleic acid may
include sequence that corresponds to both, a first site on the
target sequence that is cleaved in a first Cas9 mediated event, and
a second site on the target sequence that is cleaved in a second
Cas9 mediated event.
[0298] In certain embodiments, the template nucleic acid can
include sequence which results in an alteration in the coding
sequence of a translated sequence, e.g., one which results in the
substitution of one amino acid for another in a protein product,
e.g., transforming a mutant allele into a wild type allele,
transforming a wild type allele into a mutant allele, and/or
introducing a stop codon, insertion of an amino acid residue,
deletion of an amino acid residue, or a nonsense mutation. In
certain embodiments, the template nucleic acid can include sequence
which results in an alteration in a non-coding sequence, e.g., an
alteration in an exon or in a 5' or 3' non-translated or
non-transcribed region. Such alterations include an alteration in a
control element, e.g., a promoter, enhancer, and an alteration in a
cis-acting or trans-acting control element.
[0299] A template nucleic acid having homology with a target
position in a target gene may be used to alter the structure of a
target sequence. The template sequence may be used to alter an
unwanted structure, e.g., an unwanted or mutant nucleotide. The
template nucleic acid may include sequence which, when integrated,
results in: decreasing the activity of a positive control element;
increasing the activity of a positive control element; decreasing
the activity of a negative control element; increasing the activity
of a negative control element; decreasing the expression of a gene;
increasing the expression of a gene; increasing resistance to a
disorder or disease; increasing resistance to viral entry;
correcting a mutation or altering an unwanted amino acid residue
conferring, increasing, abolishing or decreasing a biological
property of a gene product, e.g., increasing the enzymatic activity
of an enzyme, or increasing the ability of a gene product to
interact with another molecule.
[0300] The template nucleic acid may include sequence which results
in: a change in sequence of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12
or more nucleotides of the target sequence. In an embodiment, the
template nucleic acid may be 20+/-10, 30+/-10, 40+/-10, 50+/-10,
60+/-10, 70+/-10, 80+/-10, 90+/-10, 100+/-10, 110+/-10, 120+/-10,
130+/-10, 140+/-10, 150+/-10, 160+/-10, 170+/-10, 180+/-10,
190+/-10, 200+/-10, 210+/-10, of 220+/-10 nucleotides in length. In
an embodiment, the template nucleic acid may be 30+/-20, 40+/-20,
50+/-20, 60+/-20, 70+/-20, 80+/-20, 90+/-20, 100+/-20, 110+/-20,
120+/-20, 130+/-20, 140+/-20, I 50+/-20, 160+/-20, 170+/-20,
180+/-20, 190+/-20, 200+/-20, 210+/-20, of 220+/-20 nucleotides in
length. In an embodiment, the template nucleic acid is 10 to 1,000,
20 to 900, 30 to 800, 40 to 700, 50 to 600, 50 to 500, 50 to 400,
50 to 300, 50 to 200, or 50 to 100 nucleotides in length.
[0301] A template nucleic acid comprises the following components:
[5' homology arm]-[replacement sequence]-[3' homology arm]. The
homology arms provide for recombination into the chromosome, thus
replacing the undesired element, e.g., a mutation or signature,
with the replacement sequence. In an embodiment, the homology arms
flank the most distal cleavage sites. In an embodiment, the 3' end
of the 5' homology arm is the position next to the 5' end of the
replacement sequence. In an embodiment, the 5' homology arm can
extend at least 10, 20, 30, 40, 50, 100, 200, 300, 400, 500, 600,
700, 800, 900, 1000, 1500, or 2000 nucleotides 5' from the 5' end
of the replacement sequence. In an embodiment, the 5' end of the 3'
homology arm is the position next to the 3' end of the replacement
sequence. In an embodiment, the 3' homology arm can extend at least
10, 20, 30, 40, 50, 100, 200, 300, 400, 500, 600, 700, 800, 900,
1000, 1500, or 2000 nucleotides 3' from the 3' end of the
replacement sequence.
[0302] In certain embodiments, one or both homology arms may be
shortened to avoid including certain sequence repeat elements. For
example, a 5' homology arm may be shortened to avoid a sequence
repeat element. In other embodiments, a 3' homology arm may be
shortened to avoid a sequence repeat element. In some embodiments,
both the 5' and the 3' homology arms may be shortened to avoid
including certain sequence repeat elements.
[0303] In certain embodiments, a template nucleic acids for
correcting a mutation may designed for use as a single-stranded
oligonucleotide. When using a single-stranded oligonucleotide, 5'
and 3' homology arms may range up to about 200 base pairs (bp) in
length, e.g., at least 25, 50, 75, 100, 125, 150, 175, or 200 bp in
length.
Cas9 Effector Protein Complex System Promoted Non-Homologous
End-Joining
[0304] In certain embodiments, nuclease-induced non-homologous
end-joining (NHEJ) can be used to target gene-specific knockouts.
Nuclease-induced NHEJ can also be used to remove (e.g., delete)
sequence in a gene of interest. Generally, NHEJ repairs a
double-strand break in the DNA by joining together the two ends;
however, generally, the original sequence is restored only if two
compatible ends, exactly as they were formed by the double-strand
break, are perfectly ligated. The DNA ends of the double-strand
break are frequently the subject of enzymatic processing, resulting
in the addition or removal of nucleotides, at one or both strands,
prior to rejoining of the ends. This results in the presence of
insertion and/or deletion (indel) mutations in the DNA sequence at
the site of the NHEJ repair. Two-thirds of these mutations
typically alter the reading frame and, therefore, produce a
non-functional protein. Additionally, mutations that maintain the
reading frame, but which insert or delete a significant amount of
sequence, can destroy functionality of the protein. This is locus
dependent as mutations in critical functional domains are likely
less tolerable than mutations in non-critical regions of the
protein. The indel mutations generated by NHEJ are unpredictable in
nature; however, at a given break site certain indel sequences are
favored and are over represented in the population, likely due to
small regions of microhomology. The lengths of deletions can vary
widely; most commonly in the 1-50 bp range, but they can easily be
greater than 50 bp, e.g., they can easily reach greater than about
100-200 bp. Insertions tend to be shorter and often include short
duplications of the sequence immediately surrounding the break
site. However, it is possible to obtain large insertions, and in
these cases, the inserted sequence has often been traced to other
regions of the genome or to plasmid DNA present in the cells.
[0305] Because NHEJ is a mutagenic process, it may also be used to
delete small sequence motifs as long as the generation of a
specific final sequence is not required. If a double-strand break
is targeted near to a short target sequence, the deletion mutations
caused by the NHEJ repair often span, and therefore remove, the
unwanted nucleotides. For the deletion of larger DNA segments,
introducing two double-strand breaks, one on each side of the
sequence, can result in NHEJ between the ends with removal of the
entire intervening sequence. Both of these approaches can be used
to delete specific DNA sequences; however, the error-prone nature
of NHEJ may still produce indel mutations at the site of
repair.
[0306] Both double strand cleaving Type II molecule, in particular
Cas9 or an ortholog or homolog thereof, preferably Cas9 molecules
and single strand, or nickase, Type II molecule, in particular Cas9
or an ortholog or homolog thereof, preferably Cas9 molecules can be
used in the methods and compositions described herein to generate
NHEJ-mediated indels. NHEJ-mediated indels targeted to the gene,
e.g., a coding region, e.g., an early coding region of a gene of
interest can be used to knockout (i.e., eliminate expression of) a
gene of interest. For example, early coding region of a gene of
interest includes sequence immediately following a transcription
start site, within a first exon of the coding sequence, or within
500 bp of the transcription start site (e.g., less than 500, 450,
400, 350, 300, 250, 200, 150, 100 or 50 bp).
[0307] In an embodiment, in which a guide RNA and Type II molecule,
in particular Cas9 or an ortholog or homolog thereof, preferably
Cas9 nuclease generate a double strand break for the purpose of
inducing NHEJ-mediated indels, a guide RNA may be configured to
position one double-strand break in close proximity to a nucleotide
of the target position. In an embodiment, the cleavage site may be
between 0-500 bp away from the target position (e.g., less than
500, 400, 300, 200, 100, 50, 40, 30, 25, 20, 15, 10, 9, 8, 7, 6, 5,
4, 3, 2 or 1 bp from the target position).
[0308] In an embodiment, in which two guide RNAs complexing with
Type II molecules, in particular Cas9 or an ortholog or homolog
thereof, preferably Cas9 nickases induce two single strand breaks
for the purpose of inducing NHEJ-mediated indels, two guide RNAs
may be configured to position two single-strand breaks to provide
for NHEJ repair a nucleotide of the target position.
Cas9 Effector Protein Complexes can Deliver Functional
Effectors
[0309] Unlike CRISPR-Cas-mediated gene knockout, which permanently
eliminates expression by mutating the gene at the DNA level,
CRISPR-Cas knockdown allows for temporary reduction of gene
expression through the use of artificial transcription factors.
Mutating key residues in both DNA cleavage domains of the Cas9
protein results in the generation of a catalytically inactive Cas9.
A catalytically inactive Cas9 complexes with a guide RNA and
localizes to the DNA sequence specified by that guide RNA's
targeting domain, however, it does not cleave the target DNA.
Fusion of the inactive Cas9 protein to an effector domain, e.g., a
transcription repression domain, enables recruitment of the
effector to any DNA site specified by the guide RNA. In certain
embodiments, Cas9 may be fused to a transcriptional repression
domain and recruited to the promoter region of a gene. Especially
for gene repression, it is contemplated herein that blocking the
binding site of an endogenous transcription factor would aid in
downregulating gene expression. In another embodiment, an inactive
Cas9 can be fused to a chromatin modifying protein. Altering
chromatin status can result in decreased expression of the target
gene.
[0310] In an embodiment, a guide RNA molecule can be targeted to a
known transcription response elements (e.g., promoters, enhancers,
etc.), a known upstream activating sequences, and/or sequences of
unknown or known function that are suspected of being able to
control expression of the target DNA.
[0311] In some methods, a target polynucleotide can be inactivated
to effect the modification of the expression in a cell. For
example, upon the binding of a CRISPR complex to a target sequence
in a cell, the target polynucleotide is inactivated such that the
sequence is not transcribed, the coded protein is not produced, or
the sequence does not function as the wild-type sequence does. For
example, a protein or microRNA coding sequence may be inactivated
such that the protein is not produced.
[0312] In certain embodiments, the CRISPR enzyme comprises one or
more mutations selected from the group consisting of D917A, E1006A
and D1225A and/or the one or more mutations is in a RuvC domain of
the CRISPR enzyme or is a mutation as otherwise as discussed
herein. In some embodiments, the CRISPR enzyme has one or more
mutations in a catalytic domain, wherein when transcribed, the
direct repeat sequence forms a single stem loop and the guide
sequence directs sequence-specific binding of a CRISPR complex to
the target sequence, and wherein the enzyme further comprises a
functional domain. In some embodiments, the functional domain is a
transcriptional activation domain, preferably VP64. In some
embodiments, the functional domain is a transcription repression
domain, preferably KRAB. In some embodiments, the transcription
repression domain is SID, or concatemers of SID (eg SID4X). In some
embodiments, the functional domain is an epigenetic modifying
domain, such that an epigenetic modifying enzyme is provided. In
some embodiments, the functional domain is an activation domain,
which may be the P65 activation domain.
Delivery of the CRISPR-Cas9 Complex or Components Thereof
[0313] Through this disclosure and the knowledge in the art, TALEs,
CRISPR-Cas system, specifically the novel CRISPR systems described
herein, or components thereof or nucleic acid molecules thereof
(including, for instance HDR template) or nucleic acid molecules
encoding or providing components thereof may be delivered by a
delivery system herein described both generally and in detail.
[0314] Vector delivery, e.g., plasmid, viral delivery: The CRISPR
enzyme, for instance a Cas9, and/or any of the present RNAs, for
instance a guide RNA, can be delivered using any suitable vector,
e.g., plasmid or viral vectors, such as adeno associated virus
(AAV), lentivirus, adenovirus or other viral vector types, or
combinations thereof. Cas9 and one or more guide RNAs can be
packaged into one or more vectors, e.g., plasmid or viral vectors.
In some embodiments, the vector, e.g., plasmid or viral vector is
delivered to the tissue of interest by, for example, an
intramuscular injection, while other times the delivery is via
intravenous, transdermal, intranasal, oral, mucosal, or other
delivery methods. Such delivery may be either via a single dose, or
multiple doses. One skilled in the art understands that the actual
dosage to be delivered herein may vary greatly depending upon a
variety of factors, such as the vector choice, the target cell,
organism, or tissue, the general condition of the subject to be
treated, the degree of transformation/modification sought, the
administration route, the administration mode, the type of
transformation/modification sought, etc.
[0315] Such a dosage may further contain, for example, a carrier
(water, saline, ethanol, glycerol, lactose, sucrose, calcium
phosphate, gelatin, dextran, agar, pectin, peanut oil, sesame oil,
etc.), a diluent, a pharmaceutically-acceptable carrier (e.g.,
phosphate-buffered saline), a pharmaceutically-acceptable
excipient, and/or other compounds known in the art. The dosage may
further contain one or more pharmaceutically acceptable salts such
as, for example, a mineral acid salt such as a hydrochloride, a
hydrobromide, a phosphate, a sulfate, etc.; and the salts of
organic acids such as acetates, propionates, malonates, benzoates,
etc. Additionally, auxiliary substances, such as wetting or
emulsifying agents, pH buffering substances, gels or gelling
materials, flavorings, colorants, microspheres, polymers,
suspension agents, etc. may also be present herein. In addition,
one or more other conventional pharmaceutical ingredients, such as
preservatives, humectants, suspending agents, surfactants,
antioxidants, anticaking agents, fillers, chelating agents, coating
agents, chemical stabilizers, etc. may also be present, especially
if the dosage form is a reconstitutable form. Suitable exemplary
ingredients include microcrystalline cellulose,
carboxymethylcellulose sodium, polysorbate 80, phenylethyl alcohol,
chlorobutanol, potassium sorbate, sorbic acid, sulfur dioxide,
propyl gallate, the parabens, ethyl vanillin, glycerin, phenol,
parachlorophenol, gelatin, albumin and a combination thereof. A
thorough discussion of pharmaceutically acceptable excipients is
available in REMINGTON-S PHARMACEUTICAL SCIENCES (Mack Pub. Co.,
N.J. 1991) which is incorporated by reference herein.
[0316] In an embodiment herein the delivery is via an adenovirus,
which may be at a single booster dose containing at least
1.times.10.sup.5 particles (also referred to as particle units, pu)
of adenoviral vector. In an embodiment herein, the dose preferably
is at least about 1.times.10.sup.6 particles (for example, about
1.times.10.sup.6-1.times.10.sup.12 particles), more preferably at
least about 1.times.10.sup.7 particles, more preferably at least
about 1.times.10.sup.8 particles (e.g., about
1.times.10.sup.8-1.times.10.sup.11 particles or about
1.times.10.sup.8-1.times.10.sup.12 particles), and most preferably
at least about 1.times.10.sup.0 particles (e.g., about
1.times.10.sup.9-1.times.10.sup.10 particles or about
1.times.10.sup.9-1.times.10.sup.12 particles), or even at least
about 1.times.10.sup.10 particles (e.g., about
1.times.10.sup.10-1.times.10.sup.12 particles) of the adenoviral
vector. Alternatively, the dose comprises no more than about
1.times.10.sup.14 particles, preferably no more than about
1.times.10.sup.13 particles, even more preferably no more than
about 1.times.10.sup.12 particles, even more preferably no more
than about 1.times.10.sup.11 particles, and most preferably no more
than about 1.times.10.sup.10 particles (e.g., no more than about
1.times.10.sup.9 articles). Thus, the dose may contain a single
dose of adenoviral vector with, for example, about 1.times.10.sup.6
particle units (pu), about 2.times.10.sup.6 pu, about
4.times.10.sup.6 pu, about 1.times.10.sup.7 pu, about
2.times.10.sup.7 pu, about 4.times.10.sup.7 pu, about
1.times.10.sup.8 pu, about 2.times.10.sup.8 pu, about
4.times.10.sup.8 pu, about 1.times.10.sup.9 pu, about
2.times.10.sup.9 pu, about 4.times.10.sup.9 pu, about
1.times.10.sup.10 pu, about 2.times.10.sup.10 pu, about
4.times.10.sup.10 pu, about 1.times.10.sup.11 pu, about
2.times.10.sup.11 pu, about 4.times.10.sup.11 pu, about
1.times.10.sup.12 pu, about 2.times.10.sup.12 pu, or about
4.times.10.sup.12 pu of adenoviral vector. See, for example, the
adenoviral vectors in U.S. Pat. No. 8,454,972 B2 to Nabel, et. al.,
granted on Jun. 4, 2013; incorporated by reference herein, and the
dosages at col 29, lines 36-58 thereof. In an embodiment herein,
the adenovirus is delivered via multiple doses.
[0317] In an embodiment herein, the delivery is via an AAV. A
therapeutically effective dosage for in vivo delivery of the AAV to
a human is believed to be in the range of from about 20 to about 50
ml of saline solution containing from about 1.times.10.sup.10 to
about 1.times.10.sup.10 functional AAV/ml solution. The dosage may
be adjusted to balance the therapeutic benefit against any side
effects. In an embodiment herein, the AAV dose is generally in the
range of concentrations of from about 1.times.10.sup.5 to
1.times.10.sup.50 genomes AAV, from about 1.times.10.sup.8 to
1.times.10.sup.20 genomes AAV, from about 1.times.10.sup.10 to
about 1.times.10.sup.16 genomes, or about 1.times.10.sup.11 to
about 1.times.10.sup.16 genomes AAV. A human dosage may be about
1.times.10.sup.13 genomes AAV. Such concentrations may be delivered
in from about 0.001 ml to about 100 ml, about 0.05 to about 50 ml,
or about 10 to about 25 ml of a carrier solution. Other effective
dosages can be readily established by one of ordinary skill in the
art through routine trials establishing dose response curves. See,
for example, U.S. Pat. No. 8,404,658 B2 to Hajjar, et al., granted
on Mar. 26, 2013, at col. 27, lines 45-60.
[0318] In an embodiment herein the delivery is via a plasmid. In
such plasmid compositions, the dosage should be a sufficient amount
of plasmid to elicit a response. For instance, suitable quantities
of plasmid DNA in plasmid compositions can be from about 0.1 to
about 2 mg, or from about 1 .mu.g to about 10 .mu.g per 70 kg
individual. Plasmids of the invention will generally comprise (i) a
promoter; (ii) a sequence encoding a CRISPR enzyme, operably linked
to said promoter; (iii) a selectable marker; (iv) an origin of
replication; and (v) a transcription terminator downstream of and
operably linked to (ii). The plasmid can also encode the RNA
components of a CRISPR complex, but one or more of these may
instead be encoded on a different vector.
[0319] The doses herein are based on an average 70 kg individual.
The frequency of administration is within the ambit of the medical
or veterinary practitioner (e.g., physician, veterinarian), or
scientist skilled in the art. It is also noted that mice used in
experiments are typically about 20 g and from mice experiments one
can scale up to a 70 kg individual.
[0320] In some embodiments the RNA molecules of the invention are
delivered in liposome or lipofectin formulations and the like and
can be prepared by methods well known to those skilled in the art.
Such methods are described, for example, in U.S. Pat. Nos.
5,593,972, 5,589,466, and 5,580,859, which are herein incorporated
by reference. Delivery systems aimed specifically at the enhanced
and improved delivery of siRNA into mammalian cells have been
developed, (see, for example, Shen et al FEBS Let. 2003,
539:111-114; Xia et al., Nat. Biotech. 2002, 20:1006-1010; Reich et
al., Mol. Vision. 2003, 9: 210-216; Sorensen et al., J. Mol. Biol.
2003, 327: 761-766; Lewis et al., Nat. Gen. 2002, 32: 107-108 and
Simeoni et al., NAR 2003, 31, 11: 2717-2724) and may be applied to
the present invention. siRNA has recently been successfully used
for inhibition of gene expression in primates (see for example.
Tolentino et al., Retina 24(4):660 which may also be applied to the
present invention.
[0321] Indeed, RNA delivery is a useful method of in vivo delivery.
It is possible to deliver Cas9 and gRNA (and, for instance, HR
repair template) into cells using liposomes or nanoparticles. Thus
delivery of the CRISPR enzyme, such as a Cas9 and/or delivery of
the RNAs of the invention may be in RNA form and via microvesicles,
liposomes or nanoparticles. For example, Cas9 mRNA and gRNA can be
packaged into liposomal particles for delivery in vivo. Liposomal
transfection reagents such as lipofectamine from Life Technologies
and other reagents on the market can effectively deliver RNA
molecules into the liver.
[0322] Means of delivery of RNA also preferred include delivery of
RNA via nanoparticles (Cho, S., Goldberg, M., Son, S., Xu, Q.,
Yang, F., Mei, Y., Bogatyrev, S., Langer, R. and Anderson, D.,
Lipid-like nanoparticles for small interfering RNA delivery to
endothelial cells, Advanced Functional Materials, 19: 3112-3118,
2010) or exosomes (Schroeder, A., Levins, C., Cortez, C., Langer,
R., and Anderson, D., Lipid-based nanotherapeutics for siRNA
delivery, Journal of Internal Medicine, 267: 9-21, 2010, PMID:
20059641). Indeed, exosomes have been shown to be particularly
useful in delivery siRNA, a system with some parallels to the
CRISPR system. For instance, El-Andaloussi S, et al.
("Exosome-mediated delivery of siRNA in vitro and in vivo." Nat
Protoc. 2012 December; 7(12):2112-26. doi: 10.1038/nprot.2012.131.
Epub 2012 Nov. 15.) describe how exosomes are promising tools for
drug delivery across different biological barriers and can be
harnessed for delivery of siRNA in vitro and in vivo. Their
approach is to generate targeted exosomes through transfection of
an expression vector, comprising an exosomal protein fused with a
peptide ligand. The exosomes are then purify and characterized from
transfected cell supernatant, then RNA is loaded into the exosomes.
Delivery or administration according to the invention can be
performed with exosomes, in particular but not limited to the
brain. Vitamin E (.alpha.-tocopherol) may be conjugated with CRISPR
Cas and delivered to the brain along with high density lipoprotein
(HDL), for example in a similar manner as was done by Uno et al.
(HUMAN GENE THERAPY 22:711-719 (June 2011)) for delivering
short-interfering RNA (siRNA) to the brain. Mice were infused via
Osmotic minipumps (model 1007D; Alzet, Cupertino, Calif.) filled
with phosphate-buffered saline (PBS) or free TocsiBACE or
Toc-siBACE/HDL and connected with Brain Infusion Kit 3 (Alzet). A
brain-infusion cannula was placed about 0.5 mm posterior to the
bregma at midline for infusion into the dorsal third ventricle. Uno
et al. found that as little as 3 nmol of Toc-siRNA with HDL could
induce a target reduction in comparable degree by the same ICV
infusion method. A similar dosage of CRISPR Cas9 conjugated to
.alpha.-tocopherol and co-administered with HDL targeted to the
brain may be contemplated for humans in the present invention, for
example, about 3 nmol to about 3 .mu.mol of CRISPR Cas9 targeted to
the brain may be contemplated. Zou et al. ((HUMAN GENE THERAPY
22:465-475 (April 2011)) describes a method of lentiviral-mediated
delivery of short-hairpin RNAs targeting PKC.gamma. for in vivo
gene silencing in the spinal cord of rats. Zou et al. administered
about 10 .mu.l of a recombinant lentivirus having a titer of
1.times.10.sup.9 transducing units (TU)/ml by an intrathecal
catheter. A similar dosage of CRISPR Cas9 expressed in a lentiviral
vector targeted to the brain may be contemplated for humans in the
present invention, for example, about 10-50 ml of CRISPR Cas
targeted to the brain in a lentivirus having a titer of
1.times.10.sup.9 transducing units (TU)/ml may be contemplated.
[0323] In terms of local delivery to the brain, this can be
achieved in various ways. For instance, material can be delivered
intrastriatally e.g., by injection. Injection can be performed
stereotactically via a craniotomy.
[0324] Enhancing NHEJ or HR efficiency is also helpful for
delivery. It is preferred that NHEJ efficiency is enhanced by
co-expressing end-processing enzymes such as Trex2 (Dumitrache et
al. Genetics. 2011 August; 188(4): 787-797). It is preferred that
HR efficiency is increased by transiently inhibiting NHEJ
machineries such as Ku70 and Ku86. HR efficiency can also be
increased by co-expressing prokaryotic or eukaryotic homologous
recombination enzymes such as RecBCD, RecA.
Packaging and Promoters Generally
[0325] Ways to package Cas9 coding nucleic acid molecules, e.g.,
DNA, into vectors, e.g., viral vectors, to mediate genome
modification in vivo include:
[0326] To achieve NHEJ-mediated gene knockout: [0327] Single virus
vector: [0328] Vector containing two or more expression cassettes:
[0329] Promoter-Cas9 coding nucleic acid molecule-terminator [0330]
Promoter-gRNA1-terminator [0331] Promoter-gRNA2-terminator [0332]
Promoter-gRNA(N)-terminator (up to size limit of vector) [0333]
Double virus vector: [0334] Vector 1 containing one expression
cassette for driving the expression of Cas9 Promoter-Cas9 coding
nucleic acid molecule-terminator [0335] Vector 2 containing one
more expression cassettes for driving the expression of one or more
guideRNAs [0336] Promoter-gRNA1-terminator [0337]
Promoter-gRNA(N)-terminator (up to size limit of vector)
[0338] To mediate homology-directed repair. [0339] In addition to
the single and double virus vector approaches described above, an
additional vector is used to deliver a homology-direct repair
template.
[0340] The promoter used to drive Cas9 coding nucleic acid molecule
expression can include:
[0341] AAV ITR can serve as a promoter: this is advantageous for
eliminating the need for an additional promoter element (which can
take up space in the vector). The additional space freed up can be
used to drive the expression of additional elements (gRNA, etc.).
Also, ITR activity is relatively weaker, so can be used to reduce
potential toxicity due to over expression of Cas9.
[0342] For ubiquitous expression, can use promoters: CMV, CAG, CBh,
PGK, SV40, Ferritin heavy or light chains, etc.
[0343] For brain or other CNS expression, can use promoters:
SynapsinI for all neurons, CaMKIIalpha for excitatory neurons,
GAD67 or GAD65 or VGAT for GABAergic neurons, etc.
[0344] For liver expression, can use Albumin promoter.
[0345] For lung expression, can use SP-B.
[0346] For endothelial cells, can use ICAM.
[0347] For hematopoietic cells can use IFNbeta or CD45.
[0348] For Osteoblasts can use OG-2.
[0349] The promoter used to drive guide RNA can include:
[0350] Pol III promoters such as U6 or H1
[0351] Use of Pol II promoter and intronic cassettes to express
Guide RNA
[0352] Adeno Associated Virus (AAV)
[0353] Cas9 and one or more guide RNA can be delivered using adeno
associated virus (AAV), lentivirus, adenovirus or other plasmid or
viral vector types, in particular, using formulations and doses
from, for example, U.S. Pat. No. 8,454,972 (formulations, doses for
adenovirus), U.S. Pat. No. 8,404,658 (formulations, doses for AAV)
and U.S. Pat. No. 5,846,946 (formulations, doses for DNA plasmids)
and from clinical trials and publications regarding the clinical
trials involving lentivirus, AAV and adenovirus. For examples, for
AAV, the route of administration, formulation and dose can be as in
U.S. Pat. No. 8,454,972 and as in clinical trials involving AAV.
For Adenovirus, the route of administration, formulation and dose
can be as in U.S. Pat. No. 8,404,658 and as in clinical trials
involving adenovirus. For plasmid delivery, the route of
administration, formulation and dose can be as in U.S. Pat. No.
5,846,946 and as in clinical studies involving plasmids. Doses may
be based on or extrapolated to an average 70 kg individual (e.g., a
male adult human), and can be adjusted for patients, subjects,
mammals of different weight and species. Frequency of
administration is within the ambit of the medical or veterinary
practitioner (e.g., physician, veterinarian), depending on usual
factors including the age, sex, general health, other conditions of
the patient or subject and the particular condition or symptoms
being addressed. The viral vectors can be injected into the tissue
of interest. For cell-type specific genome modification, the
expression of Cas9 can be driven by a cell-type specific promoter.
For example, liver-specific expression might use the Albumin
promoter and neuron-specific expression (e.g., for targeting CNS
disorders) might use the Synapsin I promoter.
[0354] In terms of in vivo delivery, AAV is advantageous over other
viral vectors for a couple of reasons: [0355] Low toxicity (this
may be due to the purification method not requiring ultra
centrifugation of cell particles that can activate the immune
response) and [0356] Low probability of causing insertional
mutagenesis because it doesN-t integrate into the host genome.
[0357] AAV has a packaging limit of 4.5 or 4.75 Kb. This means that
Cas9 as well as a promoter and transcription terminator have to be
all fit into the same viral vector. Constructs larger than 4.5 or
4.75 Kb will lead to significantly reduced virus production. SpCas9
is quite large, the gene itself is over 4.1 Kb, which makes it
difficult for packing into AAV. Therefore embodiments of the
invention include utilizing homologs of Cas9 that are shorter. For
example:
TABLE-US-00010 Species Cas9 Size Corynebacter diphtheriae 3252
Eubacterium ventriosum 3321 Streptococcus pasteurianus 3390
Lactobacillus farciminis 3378 Sphaerochaeta globus 3537
Azospirillum B510 3504 Gluconacetobacter diazotrophicus 3150
Neisseria cinerea 3246 Roseburia intestinalis 3420 Parvibaculum
lavamentivorans 3111 Staphylococcus aureus 3159 Nitratifractor
salsuginis DSM 16511 3396 Campylobacter lari CF89-12 3009
Streptococcus thermophilus LMD-9 3396
[0358] These species are therefore, in general, preferred Cas9
species.
[0359] As to AAV, the AAV can be AAV1, AAV2, AAV5 or any
combination thereof. One can select the AAV of the AAV with regard
to the cells to be targeted; e.g., one can select AAV serotypes 1,
2, 5 or a hybrid capsid AAV1, AAV2, AAV5 or any combination thereof
for targeting brain or neuronal cells; and one can select AAV4 for
targeting cardiac tissue. AAV8 is useful for delivery to the liver.
The herein promoters and vectors are preferred individually. A
tabulation of certain AAV serotypes as to these cells (see Grimm,
D. et al, J. Virol. 82: 5887-5911 (2008)) is as follows:
TABLE-US-00011 Cell Line AAV-1 AAV-2 AAV-3 AAV-4 AAV-5 AAV-6 AAV-8
AAV-9 Huh-7 13 100 2.5 0.0 0.1 10 0.7 0.0 HEK293 25 100 2.5 0.1 0.1
5 0.7 0.1 HeLa 3 100 2.0 0.1 6.7 1 0.2 0.1 HepG2 3 100 16.7 0.3 1.7
5 0.3 ND Hep1A 20 100 0.2 1.0 0.1 1 0.2 0.0 911 17 100 11 0.2 0.1
17 0.1 ND CHO 100 100 14 1.4 333 50 10 1.0 COS 33 100 33 3.3 5.0 14
2.0 0.5 MeWo 10 100 20 0.3 6.7 10 1.0 0.2 NIH3T3 10 100 2.9 2.9 0.3
10 0.3 ND A549 14 100 20 ND 0.5 10 0.5 0.1 HT1180 20 100 10 0.1 0.3
33 0.5 0.1 Monocytes 1111 100 ND ND 125 1429 ND ND Immature DC 2500
100 ND ND 222 2857 ND ND Mature DC 2222 100 ND ND 333 3333 ND
ND
[0360] Lentivirus
[0361] Lentiviruses are complex retroviruses that have the ability
to infect and express their genes in both mitotic and post-mitotic
cells. The most commonly known lentivirus is the human
immunodeficiency virus (HIV), which uses the envelope glycoproteins
of other viruses to target a broad range of cell types.
[0362] Lentiviruses may be prepared as follows. After cloning
pCasES10 (which contains a lentiviral transfer plasmid backbone),
HEK293FT at low passage (p=5) were seeded in a T-75 flask to 50%
confluence the day before transfection in DMEM with 10% fetal
bovine serum and without antibiotics. After 20 hours, media was
changed to OptiMEM (serum-free) media and transfection was done 4
hours later. Cells were transfected with 10 .mu.g of lentiviral
transfer plasmid (pCasES10) and the following packaging plasmids: 5
.mu.g of pMD2.G (VSV-g pseudotype), and 7.5 ug of psPAX2
(gag/pol/rev/tat). Transfection was done in 4 mL OptiMEM with a
cationic lipid delivery agent (50 uL Lipofectamine 2000 and 100 ul
Plus reagent). After 6 hours, the media was changed to
antibiotic-free DMEM with 10% fetal bovine serum. These methods use
serum during cell culture, but serum-free methods are
preferred.
[0363] Lentivirus may be purified as follows. Viral supernatants
were harvested after 48 hours. Supernatants were first cleared of
debris and filtered through a 0.45 um low protein binding (PVDF)
filter. They were then spun in a ultracentrifuge for 2 hours at
24,000 rpm. Viral pellets were resuspended in 50 ul of DMEM
overnight at 4 C. They were then aliquotted and immediately frozen
at -80.degree. C.
[0364] In another embodiment, minimal non-primate lentiviral
vectors based on the equine infectious anemia virus (EIAV) are also
contemplated, especially for ocular gene therapy (see, e.g.,
Balagaan, J Gene Med 2006; 8: 275-285). In another embodiment,
RetinoStat.RTM., an equine infectious anemia virus-based lentiviral
gene therapy vector that expresses angiostatic proteins endostatin
and angiostatin that is delivered via a subretinal injection for
the treatment of the web form of age-related macular degeneration
is also contemplated (see, e.g., Binley et al., HUMAN GENE THERAPY
23:980-991 (September 2012)) and this vector may be modified for
the CRISPR-Cas system of the present invention.
[0365] In another embodiment, self-inactivating lentiviral vectors
with an siRNA targeting a common exon shared by HIV tat/rev, a
nucleolar-localizing TAR decoy, and an anti-CCR5-specific
hammerhead ribozyme (see, e.g., DiGiusto et al. (2010) Sci Transl
Med 2:36ra43) may be used/and or adapted to the CRISPR-Cas system
of the present invention. A minimum of 2.5.times.10.sup.6 CD34+
cells per kilogram patient weight may be collected and
prestimulated for 16 to 20 hours in X-VIVO 15 medium (Lonza)
containing 2 .mu.mol/L-glutamine, stem cell factor (100 ng/ml),
Flt-3 ligand (Flt-3L) (100 ng/ml), and thrombopoietin (10 ng/ml)
(CellGenix) at a density of 2.times.10.sup.6 cells/ml.
Prestimulated cells may be transduced with lentiviral at a
multiplicity of infection of 5 for 16 to 24 hours in 75-cm.sup.2
tissue culture flasks coated with fibronectin (25 mg/cm.sup.2)
(RetroNectin, Takara Bio Inc.).
[0366] Lentiviral vectors have been disclosed as in the treatment
for ParkinsoN-s Disease, see, e.g., US Patent Publication No.
20120295960 and U.S. Pat. Nos. 7,303,910 and 7,351,585. Lentiviral
vectors have also been disclosed for the treatment of ocular
diseases, see e.g., US Patent Publication Nos. 20060281180,
20090007284, US20110117189; US20090017543; US20070054961,
US20100317109. Lentiviral vectors have also been disclosed for
delivery to the brain, see, e.g., US Patent Publication Nos.
US20110293571; US20110293571, US20040013648, US20070025970,
US20090111106 and U.S. Pat. No. 7,259,015.
RNA Delivery
[0367] RNA delivery: The CRISPR enzyme, for instance a Cas9, and/or
any of the present RNAs, for instance a guide RNA, can also be
delivered in the form of RNA. Cas9 mRNA can be generated using in
vitro transcription. For example, Cas9 mRNA can be synthesized
using a PCR cassette containing the following elements:
T7_promoter-kozak sequence (GCCACC)-Cas9-3' UTR from beta
globin-polyA tail (a string of 120 or more adenines). The cassette
can be used for transcription by T7 polymerase. Guide RNAs can also
be transcribed using in vitro transcription from a cassette
containing T7_promoter-GG-guide RNA sequence.
[0368] To enhance expression and reduce possible toxicity, the
CRISPR enzyme-coding sequence and/or the guide RNA can be modified
to include one or more modified nucleoside e.g., using pseudo-U or
5-Methyl-C.
[0369] mRNA delivery methods are especially promising for liver
delivery currently.
[0370] Much clinical work on RNA delivery has focused on RNAi or
antisense, but these systems can be adapted for delivery of RNA for
implementing the present invention. References below to RNAi etc.
should be read accordingly.
Particle Delivery Systems and/or Formulations:
[0371] Several types of particle delivery systems and/or
formulations are known to be useful in a diverse spectrum of
biomedical applications. In general, a particle is defined as a
small object that behaves as a whole unit with respect to its
transport and properties. Particles are further classified
according to diameter. Coarse particles cover a range between 2,500
and 10,000 nanometers. Fine particles are sized between 100 and
2,500 nanometers. Ultrafine particles, or nanoparticles, are
generally between 1 and 100 nanometers in size. The basis of the
100-nm limit is the fact that novel properties that differentiate
particles from the bulk material typically develop at a critical
length scale of under 100 nm.
[0372] As used herein, a particle delivery system/formulation is
defined as any biological delivery system/formulation which
includes a particle in accordance with the present invention. A
particle in accordance with the present invention is any entity
having a greatest dimension (e.g. diameter) of less than 100
microns (.mu.m). In some embodiments, inventive particles have a
greatest dimension of less than 10 .mu.m. In some embodiments,
inventive particles have a greatest dimension of less than 2000
nanometers (nm). In some embodiments, inventive particles have a
greatest dimension of less than 1000 nanometers (nm). In some
embodiments, inventive particles have a greatest dimension of less
than 900 nm, 800 nm, 700 nm, 600 nm, 500 nm, 400 nm, 300 nm, 200
nm, or 100 nm. Typically, inventive particles have a greatest
dimension (e.g., diameter) of 500 nm or less. In some embodiments,
inventive particles have a greatest dimension (e.g., diameter) of
250 nm or less. In some embodiments, inventive particles have a
greatest dimension (e.g., diameter) of 200 nm or less. In some
embodiments, inventive particles have a greatest dimension (e.g.,
diameter) of 150 nm or less. In some embodiments, inventive
particles have a greatest dimension (e.g., diameter) of 100 nm or
less. Smaller particles, e.g., having a greatest dimension of 50 nm
or less are used in some embodiments of the invention. In some
embodiments, inventive particles have a greatest dimension ranging
between 25 nm and 200 nm.
[0373] Particle characterization (including e.g., characterizing
morphology, dimension, etc.) is done using a variety of different
techniques. Common techniques are electron microscopy (TEM, SEM),
atomic force microscopy (AFM), dynamic light scattering (DLS),
X-ray photoelectron spectroscopy (XPS), powder X-ray diffraction
(XRD), Fourier transform infrared spectroscopy (FTIR),
matrix-assisted laser desorption/ionization time-of-flight mass
spectrometry (MALDI-TOF), ultraviolet-visible spectroscopy, dual
polarisation interferometry and nuclear magnetic resonance (NMR).
Characterization (dimension measurements) may be made as to native
particles (i.e., preloading) or after loading of the cargo (herein
cargo refers to e.g., one or more components of CRISPR-Cas system
e.g., CRISPR enzyme or mRNA or guide RNA, or any combination
thereof, and may include additional carriers and/or excipients) to
provide particles of an optimal size for delivery for any in vitro,
ex vivo and/or in vivo application of the present invention. In
certain preferred embodiments, particle dimension (e.g., diameter)
characterization is based on measurements using dynamic laser
scattering (DLS). Mention is made of U.S. Pat. No. 8,709,843; U.S.
Pat. No. 6,007,845; U.S. Pat. No. 5,855,913; U.S. Pat. No.
5,985,309; U.S. Pat. No. 5,543,158; and the publication by James E.
Dahlman and Carmen Barnes et al. Nature Nanotechnology (2014)
published online 11 May 2014, doi:10.1038/nnano.2014.84, concerning
particles, methods of making and using them and measurements
thereof
[0374] Particles delivery systems within the scope of the present
invention may be provided in any form, including but not limited to
solid, semi-solid, emulsion, or colloidal particles. As such any of
the delivery systems described herein, including but not limited
to, e.g., lipid-based systems, liposomes, micelles, microvesicles,
exosomes, or gene gun may be provided as particle delivery systems
within the scope of the present invention.
Particles
[0375] CRISPR enzyme mRNA and guide RNA may be delivered
simultaneously using particles or lipid envelopes; for instance,
CRISPR enzyme and RNA of the invention, e.g., as a complex, can be
delivered via a particle as in Dahlman et al., WO2015089419 A2 and
documents cited therein, such as 7C1 (see, e.g., James E. Dahlman
and Carmen Barnes et al. Nature Nanotechnology (2014) published
online 11 May 2014, doi:10.1038/nnano.2014.84), e.g., delivery
particle comprising lipid or lipidoid and hydrophilic polymer,
e.g., cationic lipid and hydrophilic polymer, for instance wherein
the cationic lipid comprises
1,2-dioleoyl-3-trimethylammonium-propane (DOTAP) or
1,2-ditetradecanoyl-sn-glycero-3-phosphocholine (DMPC) and/or
wherein the hydrophilic polymer comprises ethylene glycol or
polyethylene glycol (PEG); and/or wherein the particle further
comprises cholesterol (e.g., particle from formulation 1=DOTAP 100,
DMPC 0, PEG 0, Cholesterol 0; formulation number 2=DOTAP 90, DMPC
0, PEG 10, Cholesterol 0; formulation number 3=DOTAP 90, DMPC 0,
PEG 5, Cholesterol 5), wherein particles are formed using an
efficient, multistep process wherein first, effector protein and
RNA are mixed together, e.g., at a 1:1 molar ratio, e.g., at room
temperature, e.g., for 30 minutes, e.g., in sterile, nuclease free
1.times.PBS; and separately, DOTAP, DMPC, PEG, and cholesterol as
applicable for the formulation are dissolved in alcohol, e.g., 100%
ethanol; and, the two solutions are mixed together to form
particles containing the complexes). Nucleic acid-targeting
effector proteins (such as a Type II protein such as Cas9) mRNA and
guide RNA may be delivered simultaneously using particles or lipid
envelopes.
[0376] For example, Su X, Fricke J, Kavanagh D G, Irvine D J ("In
vitro and in vivo mRNA delivery using lipid-enveloped pH-responsive
polymer nanoparticles" Mol Pharm. 2011 Jun. 6; 8(3):774-87. doi:
10.1021/mp100390w. Epub 2011 Apr. 1) describes biodegradable
core-shell structured nanoparticles with a poly(.beta.-amino ester)
(PBAE) core enveloped by a phospholipid bilayer shell. These were
developed for in vivo mRNA delivery. The pH-responsive PBAE
component was chosen to promote endosome disruption, while the
lipid surface layer was selected to minimize toxicity of the
polycation core. Such are, therefore, preferred for delivering RNA
of the present invention.
[0377] In one embodiment, particles based on self assembling
bioadhesive polymers are contemplated, which may be applied to oral
delivery of peptides, intravenous delivery of peptides and nasal
delivery of peptides, all to the brain. Other embodiments, such as
oral absorption and ocular delivery of hydrophobic drugs are also
contemplated. The molecular envelope technology involves an
engineered polymer envelope which is protected and delivered to the
site of the disease (see, e.g., Mazza, M. et al. ACSNano, 2013.
7(2): 1016-1026; Siew, A., et al. Mol Pharm, 2012. 9(1):14-28;
Lalatsa, A., et al. J Contr Rel, 2012. 161(2):523-36; Lalatsa, A.,
et al., Mol Pharm, 2012. 9(6):1665-80; Lalatsa, A., et al. Mol
Pharm, 2012. 9(6):1764-74; Garrett, N. L., et al. J Biophotonics,
2012. 5(5-6):458-68; Garrett, N. L., et al. J Raman Spect, 2012.
43(5):681-688; Ahmad, S., et al. J Royal Soc Interface 2010.
7:S423-33; Uchegbu, I. F. Expert Opin Drug Deliv, 2006.
3(5):629-40; Qu, X., et al. Biomacromolecules, 2006. 7(12):3452-9
and Uchegbu, I. F., et al. Int J Pharm, 2001. 224:185-199). Doses
of about 5 mg/kg are contemplated, with single or multiple doses,
depending on the target tissue.
[0378] In one embodiment, particles that can deliver RNA to a
cancer cell to stop tumor growth developed by Dan Anderson's lab at
MIT may be used/and or adapted to the CRISPR Cas system of the
present invention. In particular, the Anderson lab developed fully
automated, combinatorial systems for the synthesis, purification,
characterization, and formulation of new biomaterials and
nanoformulations. See, e.g., Alabi et al., Proc Natl Acad Sci USA.
2013 Aug. 6; 110(32):12881-6; Zhang et al., Adv Mater. 2013 Sep. 6;
25(33):4641-5; Jiang et al., Nano Lett. 2013 Mar. 13;
13(3):1059-64; Karagiannis et al., ACS Nano. 2012 Oct. 23;
6(10):8484-7; Whitehead et al., ACS Nano. 2012 Aug. 28; 6(8):6922-9
and Lee et al., Nat Nanotechnol. 2012 Jun. 3; 7(6):389-93.
[0379] US patent application 20110293703 relates to lipidoid
compounds are also particularly useful in the administration of
polynucleotides, which may be applied to deliver the CRISPR Cas
system of the present invention. In one aspect, the aminoalcohol
lipidoid compounds are combined with an agent to be delivered to a
cell or a subject to form microparticles, nanoparticles, liposomes,
or micelles. The agent to be delivered by the particles, liposomes,
or micelles may be in the form of a gas, liquid, or solid, and the
agent may be a polynucleotide, protein, peptide, or small molecule.
The aminoalcohol lipidoid compounds may be combined with other
aminoalcohol lipidoid compounds, polymers (synthetic or natural),
surfactants, cholesterol, carbohydrates, proteins, lipids, etc. to
form the particles. These particles may then optionally be combined
with a pharmaceutical excipient to form a pharmaceutical
composition.
[0380] US Patent Publication No. 20110293703 also provides methods
of preparing the aminoalcohol lipidoid compounds. One or more
equivalents of an amine are allowed to react with one or more
equivalents of an epoxide-terminated compound under suitable
conditions to form an aminoalcohol lipidoid compound of the present
invention. In certain embodiments, all the amino groups of the
amine are fully reacted with the epoxide-terminated compound to
form tertiary amines. In other embodiments, all the amino groups of
the amine are not fully reacted with the epoxide-terminated
compound to form tertiary amines thereby resulting in primary or
secondary amines in the aminoalcohol lipidoid compound. These
primary or secondary amines are left as is or may be reacted with
another electrophile such as a different epoxide-terminated
compound. As will be appreciated by one skilled in the art,
reacting an amine with less than excess of epoxide-terminated
compound will result in a plurality of different aminoalcohol
lipidoid compounds with various numbers of tails. Certain amines
may be fully functionalized with two epoxide-derived compound tails
while other molecules will not be completely functionalized with
epoxide-derived compound tails. For example, a diamine or polyamine
may include one, two, three, or four epoxide-derived compound tails
off the various amino moieties of the molecule resulting in
primary, secondary, and tertiary amines. In certain embodiments,
all the amino groups are not fully functionalized. In certain
embodiments, two of the same types of epoxide-terminated compounds
are used. In other embodiments, two or more different
epoxide-terminated compounds are used. The synthesis of the
aminoalcohol lipidoid compounds is performed with or without
solvent, and the synthesis may be performed at higher temperatures
ranging from 30-100.degree. C., preferably at approximately
50-90.degree. C. The prepared aminoalcohol lipidoid compounds may
be optionally purified. For example, the mixture of aminoalcohol
lipidoid compounds may be purified to yield an aminoalcohol
lipidoid compound with a particular number of epoxide-derived
compound tails. Or the mixture may be purified to yield a
particular stereo- or regioisomer. The aminoalcohol lipidoid
compounds may also be alkylated using an alkyl halide (e.g., methyl
iodide) or other alkylating agent, and/or they may be acylated.
[0381] US Patent Publication No. 20110293703 also provides
libraries of aminoalcohol lipidoid compounds prepared by the
inventive methods. These aminoalcohol lipidoid compounds may be
prepared and/or screened using high-throughput techniques involving
liquid handlers, robots, microtiter plates, computers, etc. In
certain embodiments, the aminoalcohol lipidoid compounds are
screened for their ability to transfect polynucleotides or other
agents (e.g., proteins, peptides, small molecules) into the
cell.
[0382] US Patent Publication No. 20130302401 relates to a class of
poly(beta-amino alcohols) (PBAAs) has been prepared using
combinatorial polymerization. The inventive PBAAs may be used in
biotechnology and biomedical applications as coatings (such as
coatings of films or multilayer films for medical devices or
implants), additives, materials, excipients, non-biofouling agents,
micropatterning agents, and cellular encapsulation agents. When
used as surface coatings, these PBAAs elicited different levels of
inflammation, both in vitro and in vivo, depending on their
chemical structures. The large chemical diversity of this class of
materials allowed us to identify polymer coatings that inhibit
macrophage activation in vitro. Furthermore, these coatings reduce
the recruitment of inflammatory cells, and reduce fibrosis,
following the subcutaneous implantation of carboxylated polystyrene
microparticles. These polymers may be used to form polyelectrolyte
complex capsules for cell encapsulation. The invention may also
have many other biological applications such as antimicrobial
coatings, DNA or siRNA delivery, and stem cell tissue engineering.
The teachings of US Patent Publication No. 20130302401 may be
applied to the CRISPR Cas system of the present invention.
[0383] In another embodiment, lipid nanoparticles (LNPs) are
contemplated. An antitransthyretin small interfering RNA has been
encapsulated in lipid nanoparticles and delivered to humans (see,
e.g., Coelho et al., N Engl J Med 2013; 369:819-29), and such a
system may be adapted and applied to the CRISPR Cas system of the
present invention. Doses of about 0.01 to about 1 mg per kg of body
weight administered intravenously are contemplated. Medications to
reduce the risk of infusion-related reactions are contemplated,
such as dexamethasone, acetaminophen, diphenhydramine or
cetirizine, and ranitidine are contemplated. Multiple doses of
about 0.3 mg per kilogram every 4 weeks for five doses are also
contemplated.
[0384] LNPs have been shown to be highly effective in delivering
siRNAs to the liver (see, e.g., Tabernero et al., Cancer Discovery,
April 2013, Vol. 3, No. 4, pages 363-470) and are therefore
contemplated for delivering RNA encoding CRISPR Cas to the liver. A
dosage of about four doses of 6 mg/kg of the LNP every two weeks
may be contemplated. Tabernero et al. demonstrated that tumor
regression was observed after the first 2 cycles of LNPs dosed at
0.7 mg/kg, and by the end of 6 cycles the patient had achieved a
partial response with complete regression of the lymph node
metastasis and substantial shrinkage of the liver tumors. A
complete response was obtained after 40 doses in this patient, who
has remained in remission and completed treatment after receiving
doses over 26 months. Two patients with RCC and extrahepatic sites
of disease including kidney, lung, and lymph nodes that were
progressing following prior therapy with VEGF pathway inhibitors
had stable disease at all sites for approximately 8 to 12 months,
and a patient with PNET and liver metastases continued on the
extension study for 18 months (36 doses) with stable disease.
[0385] However, the charge of the LNP must be taken into
consideration. As cationic lipids combined with negatively charged
lipids to induce nonbilayer structures that facilitate
intracellular delivery. Because charged LNPs are rapidly cleared
from circulation following intravenous injection, ionizable
cationic lipids with pKa values below 7 were developed (see, e.g.,
Rosin et al, Molecular Therapy, vol. 19, no. 12, pages 1286-2200,
December 2011). Negatively charged polymers such as RNA may be
loaded into LNPs at low pH values (e.g., pH 4) where the ionizable
lipids display a positive charge. However, at physiological pH
values, the LNPs exhibit a low surface charge compatible with
longer circulation times. Four species of ionizable cationic lipids
have been focused upon, namely
1,2-dilineoyl-3-dimethylammonium-propane (DLinDAP),
1,2-dilinoleyloxy-3-N,N-dimethylaminopropane (DLinDMA),
1,2-dilinoleyloxy-keto-N,N-dimethyl-3-aminopropane (DLinKDMA), and
1,2-dilinoleyl-4-(2-dimethylaminoethyl)-[1,3]-dioxolane
(DLinKC2-DMA). It has been shown that LNP siRNA systems containing
these lipids exhibit remarkably different gene silencing properties
in hepatocytes in vivo, with potencies varying according to the
series DLinKC2-DMA>DLinKDMA>DLinDMA>>DLinDAP employing
a Factor VII gene silencing model (see, e.g., Rosin et al,
Molecular Therapy, vol. 19, no. 12, pages 1286-2200, December
2011). A dosage of 1 .mu.g/ml of LNP or CRISPR-Cas RNA in or
associated with the LNP may be contemplated, especially for a
formulation containing DLinKC2-DMA.
[0386] Preparation of LNPs and CRISPR Cas encapsulation may be
used/and or adapted from Rosin et al, Molecular Therapy, vol. 19,
no. 12, pages 1286-2200, December 2011). The cationic lipids
1,2-dilineoyl-3-dimethylammonium-propane (DLinDAP),
1,2-dilinoleyloxy-3-N,N-dimethylaminopropane (DLinDMA),
1,2-dilinoleyloxyketo-N,N-dimethyl-3-aminopropane (DLinK-DMA),
1,2-dilinoleyl-4-(2-dimethylaminoethyl)[1,3]-dioxolane
(DLinKC2-DMA), (3-o-[2''-(methoxypolyethyleneglycol 2000)
succinoyl]-1,2-dimyristoyl-sn-glycol (PEG-S-DMG), and
R-3-[(.omega.-methoxy-poly(ethylene glycol)2000)
carbamoyl]-1,2-dimyristyloxlpropyl-3-amine (PEG-C-DOMG) may be
provided by Tekmira Pharmaceuticals (Vancouver, Canada) or
synthesized. Cholesterol may be purchased from Sigma (St Louis,
Mo.). The specific CRISPR Cas RNA may be encapsulated in LNPs
containing DLinDAP, DLinDMA, DLinK-DMA, and DLinKC2-DMA (cationic
lipid:DSPC:CHOL: PEGS-DMG or PEG-C-DOMG at 40:10:40:10 molar
ratios). When required, 0.2% SP-DiOC18 (Invitrogen, Burlington,
Canada) may be incorporated to assess cellular uptake,
intracellular delivery, and biodistribution. Encapsulation may be
performed by dissolving lipid mixtures comprised of cationic
lipid:DSPC:cholesterol:PEG-c-DOMG (40:10:40:10 molar ratio) in
ethanol to a final lipid concentration of 10 mmol/1. This ethanol
solution of lipid may be added drop-wise to 50 mmol/1 citrate, pH
4.0 to form multilamellar vesicles to produce a final concentration
of 30% ethanol vol/vol. Large unilamellar vesicles may be formed
following extrusion of multilamellar vesicles through two stacked
80 nm Nuclepore polycarbonate filters using the Extruder (Northern
Lipids, Vancouver, Canada). Encapsulation may be achieved by adding
RNA dissolved at 2 mg/ml in 50 mmol/1 citrate, pH 4.0 containing
30% ethanol vol/vol drop-wise to extruded preformed large
unilamellar vesicles and incubation at 31.degree. C. for 30 minutes
with constant mixing to a final RNA/lipid weight ratio of 0.06/1
wt/wt. Removal of ethanol and neutralization of formulation buffer
were performed by dialysis against phosphate-buffered saline (PBS),
pH 7.4 for 16 hours using Spectra/Por 2 regenerated cellulose
dialysis membranes. Nanoparticle size distribution may be
determined by dynamic light scattering using a NICOMP 370 particle
sizer, the vesicle/intensity modes, and Gaussian fitting (Nicomp
Particle Sizing, Santa Barbara, Calif.). The particle size for all
three LNP systems may be .about.70 nm in diameter. RNA
encapsulation efficiency may be determined by removal of free RNA
using VivaPureD MiniH columns (Sartorius Stedim Biotech) from
samples collected before and after dialysis. The encapsulated RNA
may be extracted from the eluted nanoparticles and quantified at
260 nm. RNA to lipid ratio was determined by measurement of
cholesterol content in vesicles using the Cholesterol E enzymatic
assay from Wako Chemicals USA (Richmond, Va.). In conjunction with
the herein discussion of LNPs and PEG lipids, PEGylated liposomes
or LNPs are likewise suitable for delivery of a CRISPR-Cas system
or components thereof
[0387] Preparation of large LNPs may be used/and or adapted from
Rosin et al, Molecular Therapy, vol. 19, no. 12, pages 1286-2200,
December 2011. A lipid premix solution (20.4 mg/ml total lipid
concentration) may be prepared in ethanol containing DLinKC2-DMA,
DSPC, and cholesterol at 50:10:38.5 molar ratios. Sodium acetate
may be added to the lipid premix at a molar ratio of 0.75:1 (sodium
acetate:DLinKC2-DMA). The lipids may be subsequently hydrated by
combining the mixture with 1.85 volumes of citrate buffer (10
mmol/1, pH 3.0) with vigorous stirring, resulting in spontaneous
liposome formation in aqueous buffer containing 35% ethanol. The
liposome solution may be incubated at 37.degree. C. to allow for
time-dependent increase in particle size. Aliquots may be removed
at various times during incubation to investigate changes in
liposome size by dynamic light scattering (Zetasizer Nano ZS,
Malvern Instruments, Worcestershire, UK). Once the desired particle
size is achieved, an aqueous PEG lipid solution (stock=10 mg/ml
PEG-DMG in 35% (vol/vol) ethanol) may be added to the liposome
mixture to yield a final PEG molar concentration of 3.5% of total
lipid. Upon addition of PEG-lipids, the liposomes should their
size, effectively quenching further growth. RNA may then be added
to the empty liposomes at an RNA to total lipid ratio of
approximately 1:10 (wt:wt), followed by incubation for 30 minutes
at 37.degree. C. to form loaded LNPs. The mixture may be
subsequently dialyzed overnight in PBS and filtered with a
0.45-.mu.m syringe filter.
[0388] Spherical Nucleic Acid (SNA.TM.) constructs and other
nanoparticles (particularly gold nanoparticles) are also
contemplated as a means to delivery CRISPR-Cas system to intended
targets. Significant data show that AuraSense Therapeutics'
Spherical Nucleic Acid (SNA.TM.) constructs, based upon nucleic
acid-functionalized gold nanoparticles, are useful.
[0389] Literature that may be employed in conjunction with herein
teachings include: Cutler et al., J. Am. Chem. Soc. 2011
133:9254-9257, Hao et al., Small. 2011 7:3158-3162, Zhang et al.,
ACS Nano. 2011 5:6962-6970, Cutler et al., J. Am. Chem. Soc. 2012
134:1376-1391, Young et al., Nano Lett. 2012 12:3867-71, Zheng et
al., Proc. Natl. Acad. Sci. USA. 2012 109:11975-80, Mirkin,
Nanomedicine 2012 7:635-638 Zhang et al., J. Am. Chem. Soc. 2012
134:16488-1691, Weintraub, Nature 2013 495:S14-S16, Choi et al.,
Proc. Natl. Acad. Sci. USA. 2013 110(19):7625-7630, Jensen et al.,
Sci. Transl. Med. 5, 209ra152 (2013) and Mirkin, et al., Small,
10:186-192.
[0390] Self-assembling nanoparticles with RNA may be constructed
with polyethyleneimine (PEI) that is PEGylated with an Arg-Gly-Asp
(RGD) peptide ligand attached at the distal end of the polyethylene
glycol (PEG). This system has been used, for example, as a means to
target tumor neovasculature expressing integrins and deliver siRNA
inhibiting vascular endothelial growth factor receptor-2 (VEGF R2)
expression and thereby achieve tumor angiogenesis (see, e.g.,
Schiffelers et al., Nucleic Acids Research, 2004, Vol. 32, No. 19).
Nanoplexes may be prepared by mixing equal volumes of aqueous
solutions of cationic polymer and nucleic acid to give a net molar
excess of ionizable nitrogen (polymer) to phosphate (nucleic acid)
over the range of 2 to 6. The electrostatic interactions between
cationic polymers and nucleic acid resulted in the formation of
polyplexes with average particle size distribution of about 100 nm,
hence referred to here as nanoplexes. A dosage of about 100 to 200
mg of CRISPR Cas9 is envisioned for delivery in the self-assembling
nanoparticles of Schiffelers et al.
[0391] The nanoplexes of Bartlett et al. (PNAS, Sep. 25, 2007, vol.
104, no. 39) may also be applied to the present invention. The
nanoplexes of Bartlett et al. are prepared by mixing equal volumes
of aqueous solutions of cationic polymer and nucleic acid to give a
net molar excess of ionizable nitrogen (polymer) to phosphate
(nucleic acid) over the range of 2 to 6. The electrostatic
interactions between cationic polymers and nucleic acid resulted in
the formation of polyplexes with average particle size distribution
of about 100 nm, hence referred to here as nanoplexes. The
DOTA-siRNA of Bartlett et al. was synthesized as follows:
1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid
mono(N-hydroxysuccinimide ester) (DOTA-NHSester) was ordered from
Macrocyclics (Dallas, Tex.). The amine modified RNA sense strand
with a 100-fold molar excess of DOTA-NHS-ester in carbonate buffer
(pH 9) was added to a microcentrifuge tube. The contents were
reacted by stirring for 4 h at room temperature. The DOTA-RNAsense
conjugate was ethanol-precipitated, resuspended in water, and
annealed to the unmodified antisense strand to yield DOTA-siRNA.
All liquids were pretreated with Chelex-100 (Bio-Rad, Hercules,
Calif.) to remove trace metal contaminants. Tf-targeted and
nontargeted siRNA nanoparticles may be formed by using
cyclodextrin-containing polycations. Typically, nanoparticles were
formed in water at a charge ratio of 3 (+/-) and an siRNA
concentration of 0.5 g/liter. One percent of the adamantane-PEG
molecules on the surface of the targeted nanoparticles were
modified with Tf (adamantane-PEG-Tf). The nanoparticles were
suspended in a 5% (wt/vol) glucose carrier solution for
injection.
[0392] Davis et al. (Nature, Vol 464, 15 Apr. 2010) conducts a RNA
clinical trial that uses a targeted nanoparticle-delivery system
(clinical trial registration number NCT00689065). Patients with
solid cancers refractory to standard-of-care therapies are
administered doses of targeted nanoparticles on days 1, 3, 8 and 10
of a 21-day cycle by a 30-min intravenous infusion. The
nanoparticles comprise, consist essentially of, or consist of a
synthetic delivery system containing: (1) a linear,
cyclodextrin-based polymer (CDP), (2) a human transferrin protein
(TF) targeting ligand displayed on the exterior of the nanoparticle
to engage TF receptors (TFR) on the surface of the cancer cells,
(3) a hydrophilic polymer (polyethylene glycol (PEG) used to
promote nanoparticle stability in biological fluids), and (4) siRNA
designed to reduce the expression of the RRM2 (sequence used in the
clinic was previously denoted siR2B+5). The TFR has long been known
to be upregulated in malignant cells, and RRM2 is an established
anticancer target. These nanoparticles (clinical version denoted as
CALAA-01) have been shown to be well tolerated in multi-dosing
studies in non-human primates. Although a single patient with
chronic myeloid leukaemia has been administered siRNA by liposomal
delivery, Davis et al.'s clinical trial is the initial human trial
to systemically deliver siRNA with a targeted delivery system and
to treat patients with solid cancer. To ascertain whether the
targeted delivery system can provide effective delivery of
functional siRNA to human tumors, Davis et al. investigated
biopsies from three patients from three different dosing cohorts;
patients A, B and C, all of whom had metastatic melanoma and
received CALAA-01 doses of 18, 24 and 30 mg m.sup.-2 siRNA,
respectively. Similar doses may also be contemplated for the CRISPR
Cas system of the present invention. The delivery of the invention
may be achieved with nanoparticles containing a linear,
cyclodextrin-based polymer (CDP), a human transferrin protein (TF)
targeting ligand displayed on the exterior of the nanoparticle to
engage TF receptors (TFR) on the surface of the cancer cells and/or
a hydrophilic polymer (for example, polyethylene glycol (PEG) used
to promote nanoparticle stability in biological fluids).
[0393] In terms of this invention, it is preferred to have one or
more components of CRISPR complex, e.g., CRISPR enzyme or mRNA or
guide RNA delivered using nanoparticles or lipid envelopes. Other
delivery systems or vectors are may be used in conjunction with the
nanoparticle aspects of the invention.
[0394] In general, a "nanoparticle" refers to any particle having a
diameter of less than 1000 nm. In certain preferred embodiments,
nanoparticles of the invention have a greatest dimension (e.g.,
diameter) of 500 nm or less. In other preferred embodiments,
nanoparticles of the invention have a greatest dimension ranging
between 25 nm and 200 nm. In other preferred embodiments,
nanoparticles of the invention have a greatest dimension of 100 nm
or less. In other preferred embodiments, nanoparticles of the
invention have a greatest dimension ranging between 35 nm and 60
nm.
[0395] Nanoparticles encompassed in the present invention may be
provided in different forms, e.g., as solid nanoparticles (e.g.,
metal such as silver, gold, iron, titanium), non-metal, lipid-based
solids, polymers), suspensions of nanoparticles, or combinations
thereof. Metal, dielectric, and semiconductor nanoparticles may be
prepared, as well as hybrid structures (e.g., core-shell
nanoparticles). Nanoparticles made of semiconducting material may
also be labeled quantum dots if they are small enough (typically
sub 10 nm) that quantization of electronic energy levels occurs.
Such nanoscale particles are used in biomedical applications as
drug carriers or imaging agents and may be adapted for similar
purposes in the present invention.
[0396] Semi-solid and soft nanoparticles have been manufactured,
and are within the scope of the present invention. A prototype
nanoparticle of semi-solid nature is the liposome. Various types of
liposome nanoparticles are currently used clinically as delivery
systems for anticancer drugs and vaccines. Nanoparticles with one
half hydrophilic and the other half hydrophobic are termed Janus
particles and are particularly effective for stabilizing emulsions.
They can self-assemble at water/oil interfaces and act as solid
surfactants.
[0397] U.S. Pat. No. 8,709,843, incorporated herein by reference,
provides a drug delivery system for targeted delivery of
therapeutic agent-containing particles to tissues, cells, and
intracellular compartments. The invention provides targeted
particles comprising polymer conjugated to a surfactant,
hydrophilic polymer or lipid.
[0398] U.S. Pat. No. 6,007,845, incorporated herein by reference,
provides particles which have a core of a multiblock copolymer
formed by covalently linking a multifunctional compound with one or
more hydrophobic polymers and one or more hydrophilic polymers, and
contain a biologically active material.
[0399] U.S. Pat. No. 5,855,913, incorporated herein by reference,
provides a particulate composition having aerodynamically light
particles having a tap density of less than 0.4 g/cm3 with a mean
diameter of between 5 .mu.m and 30 .mu.m, incorporating a
surfactant on the surface thereof for drug delivery to the
pulmonary system.
[0400] U.S. Pat. No. 5,985,309, incorporated herein by reference,
provides particles incorporating a surfactant and/or a hydrophilic
or hydrophobic complex of a positively or negatively charged
therapeutic or diagnostic agent and a charged molecule of opposite
charge for delivery to the pulmonary system.
[0401] U.S. Pat. No. 5,543,158, incorporated herein by reference,
provides biodegradable injectable nanoparticles having a
biodegradable solid core containing a biologically active material
and poly(alkylene glycol) moieties on the surface.
[0402] WO2012135025 (also published as US20120251560), incorporated
herein by reference, describes conjugated polyethyleneimine (PEI)
polymers and conjugated aza-macrocycles (collectively referred to
as "conjugated lipomer" or "lipomers"). In certain embodiments, it
can envisioned that such conjugated lipomers can be used in the
context of the CRISPR-Cas system to achieve in vitro, ex vivo and
in vivo genomic perturbations to modify gene expression, including
modulation of protein expression.
[0403] In one embodiment, the nanoparticle may be epoxide-modified
lipid-polymer, advantageously 7C1 (see, e.g., James E. Dahlman and
Carmen Barnes et al. Nature Nanotechnology (2014) published online
11 May 2014, doi:10.1038/nnano.2014.84). C71 was synthesized by
reacting C15 epoxide-terminated lipids with PEI600 at a 14:1 molar
ratio, and was formulated with C14PEG2000 to produce nanoparticles
(diameter between 35 and 60 nm) that were stable in PBS solution
for at least 40 days.
[0404] An epoxide-modified lipid-polymer may be utilized to deliver
the CRISPR-Cas system of the present invention to pulmonary,
cardiovascular or renal cells, however, one of skill in the art may
adapt the system to deliver to other target organs. Dosage ranging
from about 0.05 to about 0.6 mg/kg are envisioned. Dosages over
several days or weeks are also envisioned, with a total dosage of
about 2 mg/kg.
Exosomes
[0405] Exosomes are endogenous nano-vesicles that transport RNAs
and proteins, and which can deliver RNA to the brain and other
target organs. To reduce immunogenicity, Alvarez-Erviti et al.
(2011, Nat Biotechnol 29: 341) used self-derived dendritic cells
for exosome production. Targeting to the brain was achieved by
engineering the dendritic cells to express Lamp2b, an exosomal
membrane protein, fused to the neuron-specific RVG peptide.
Purified exosomes were loaded with exogenous RNA by
electroporation. Intravenously injected RVG-targeted exosomes
delivered GAPDH siRNA specifically to neurons, microglia,
oligodendrocytes in the brain, resulting in a specific gene
knockdown. Pre-exposure to RVG exosomes did not attenuate
knockdown, and non-specific uptake in other tissues was not
observed. The therapeutic potential of exosome-mediated siRNA
delivery was demonstrated by the strong mRNA (60%) and protein
(62%) knockdown of BACE1, a therapeutic target in Alzheimer's
disease.
[0406] To obtain a pool of immunologically inert exosomes,
Alvarez-Erviti et al. harvested bone marrow from inbred C57BL/6
mice with a homogenous major histocompatibility complex (MHC)
haplotype. As immature dendritic cells produce large quantities of
exosomes devoid of T-cell activators such as MHC-II and CD86,
Alvarez-Erviti et al. selected for dendritic cells with
granulocyte/macrophage-colony stimulating factor (GM-CSF) for 7 d.
Exosomes were purified from the culture supernatant the following
day using well-established ultracentrifugation protocols. The
exosomes produced were physically homogenous, with a size
distribution peaking at 80 nm in diameter as determined by
nanoparticle tracking analysis (NTA) and electron microscopy.
Alvarez-Erviti et al. obtained 6-12 .mu.g of exosomes (measured
based on protein concentration) per 10.sup.6 cells.
[0407] Next, Alvarez-Erviti et al. investigated the possibility of
loading modified exosomes with exogenous cargoes using
electroporation protocols adapted for nanoscale applications. As
electroporation for membrane particles at the nanometer scale is
not well-characterized, nonspecific Cy5-labeled RNA was used for
the empirical optimization of the electroporation protocol. The
amount of encapsulated RNA was assayed after ultracentrifugation
and lysis of exosomes. Electroporation at 400 V and 125 .mu.F
resulted in the greatest retention of RNA and was used for all
subsequent experiments.
[0408] Alvarez-Erviti et al. administered 150 .mu.g of each BACE1
siRNA encapsulated in 150 .mu.g of RVG exosomes to normal C57BL/6
mice and compared the knockdown efficiency to four controls:
untreated mice, mice injected with RVG exosomes only, mice injected
with BACE1 siRNA complexed to an in vivo cationic liposome reagent
and mice injected with BACE1 siRNA complexed to RVG-9R, the RVG
peptide conjugated to 9 D-arginines that electrostatically binds to
the siRNA. Cortical tissue samples were analyzed 3 d after
administration and a significant protein knockdown (45%, P<0.05,
versus 62%, P<0.01) in both siRNA-RVG-9R-treated and siRNARVG
exosome-treated mice was observed, resulting from a significant
decrease in BACE1 mRNA levels (66% [+ or -] 15%, P<0.001 and 61%
[+ or -] 13% respectively, P<0.01). Moreover, Applicants
demonstrated a significant decrease (55%, P<0.05) in the total
[beta]-amyloid 1-42 levels, a main component of the amyloid plaques
in Alzheimer's pathology, in the RVG-exosome-treated animals. The
decrease observed was greater than the .beta.-amyloid 1-40 decrease
demonstrated in normal mice after intraventricular injection of
BACE1 inhibitors. Alvarez-Erviti et al. carried out 5'-rapid
amplification of cDNA ends (RACE) on BACE1 cleavage product, which
provided evidence of RNAi-mediated knockdown by the siRNA.
[0409] Finally, Alvarez-Erviti et al. investigated whether RNA-RVG
exosomes induced immune responses in vivo by assessing IL-6, IP-10,
TNF.alpha. and IFN-.alpha. serum concentrations. Following exosome
treatment, nonsignificant changes in all cytokines were registered
similar to siRNA-transfection reagent treatment in contrast to
siRNA-RVG-9R, which potently stimulated IL-6 secretion, confirming
the immunologically inert profile of the exosome treatment. Given
that exosomes encapsulate only 20% of siRNA, delivery with
RVG-exosome appears to be more efficient than RVG-9R delivery as
comparable mRNA knockdown and greater protein knockdown was
achieved with fivefold less siRNA without the corresponding level
of immune stimulation. This experiment demonstrated the therapeutic
potential of RVG-exosome technology, which is potentially suited
for long-term silencing of genes related to neurodegenerative
diseases. The exosome delivery system of Alvarez-Erviti et al. may
be applied to deliver the CRISPR-Cas system of the present
invention to therapeutic targets, especially neurodegenerative
diseases. A dosage of about 100 to 1000 mg of CRISPR Cas9
encapsulated in about 100 to 1000 mg of RVG exosomes may be
contemplated for the present invention.
[0410] El-Andaloussi et al. (Nature Protocols 7, 2112-2126(2012))
discloses how exosomes derived from cultured cells can be harnessed
for delivery of RNA in vitro and in vivo. This protocol first
describes the generation of targeted exosomes through transfection
of an expression vector, comprising an exosomal protein fused with
a peptide ligand. Next, El-Andaloussi et al. explain how to purify
and characterize exosomes from transfected cell supernatant. Next,
El-Andaloussi et al. detail crucial steps for loading RNA into
exosomes. Finally, El-Andaloussi et al. outline how to use exosomes
to efficiently deliver RNA in vitro and in vivo in mouse brain.
Examples of anticipated results in which exosome-mediated RNA
delivery is evaluated by functional assays and imaging are also
provided. The entire protocol takes .about.3 weeks. Delivery or
administration according to the invention may be performed using
exosomes produced from self-derived dendritic cells. From the
herein teachings, this can be employed in the practice of the
invention
[0411] In another embodiment, the plasma exosomes of Wahlgren et
al. (Nucleic Acids Research, 2012, Vol. 40, No. 17 e130) are
contemplated. Exosomes are nano-sized vesicles (30-90 nm in size)
produced by many cell types, including dendritic cells (DC), B
cells, T cells, mast cells, epithelial cells and tumor cells. These
vesicles are formed by inward budding of late endosomes and are
then released to the extracellular environment upon fusion with the
plasma membrane. Because exosomes naturally carry RNA between
cells, this property may be useful in gene therapy, and from this
disclosure can be employed in the practice of the instant
invention.
[0412] Exosomes from plasma can be prepared by centrifugation of
buffy coat at 900 g for 20 min to isolate the plasma followed by
harvesting cell supernatants, centrifuging at 300 g for 10 min to
eliminate cells and at 16 500 g for 30 min followed by filtration
through a 0.22 mm filter. Exosomes are pelleted by
ultracentrifugation at 120 000 g for 70 min. Chemical transfection
of siRNA into exosomes is carried out according to the
manufacturer's instructions in RNAi Human/Mouse Starter Kit
(Quiagen, Hilden, Germany). siRNA is added to 100 ml PBS at a final
concentration of 2 mmol/ml. After adding HiPerFect transfection
reagent, the mixture is incubated for 10 min at RT. In order to
remove the excess of micelles, the exosomes are re-isolated using
aldehyde/sulfate latex beads. The chemical transfection of CRISPR
Cas9 into exosomes may be conducted similarly to siRNA. The
exosomes may be co-cultured with monocytes and lymphocytes isolated
from the peripheral blood of healthy donors. Therefore, it may be
contemplated that exosomes containing CRISPR Cas9 may be introduced
to monocytes and lymphocytes of and autologously reintroduced into
a human. Accordingly, delivery or administration according to the
invention may be performed using plasma exosomes.
Liposomes
[0413] Delivery or administration according to the invention can be
performed with liposomes. Liposomes are spherical vesicle
structures composed of a uni- or multilamellar lipid bilayer
surrounding internal aqueous compartments and a relatively
impermeable outer lipophilic phospholipid bilayer. Liposomes have
gained considerable attention as drug delivery carriers because
they are biocompatible, nontoxic, can deliver both hydrophilic and
lipophilic drug molecules, protect their cargo from degradation by
plasma enzymes, and transport their load across biological
membranes and the blood brain barrier (BBB) (see, e.g., Spuch and
Navarro, Journal of Drug Delivery, vol. 2011, Article ID 469679, 12
pages, 2011. doi:10.1155/2011/469679 for review).
[0414] Liposomes can be made from several different types of
lipids; however, phospholipids are most commonly used to generate
liposomes as drug carriers. Although liposome formation is
spontaneous when a lipid film is mixed with an aqueous solution, it
can also be expedited by applying force in the form of shaking by
using a homogenizer, sonicator, or an extrusion apparatus (see,
e.g., Spuch and Navarro, Journal of Drug Delivery, vol. 2011,
Article ID 469679, 12 pages, 2011. doi:10.1155/2011/469679 for
review).
[0415] Several other additives may be added to liposomes in order
to modify their structure and properties. For instance, either
cholesterol or sphingomyelin may be added to the liposomal mixture
in order to help stabilize the liposomal structure and to prevent
the leakage of the liposomal inner cargo. Further, liposomes are
prepared from hydrogenated egg phosphatidylcholine or egg
phosphatidylcholine, cholesterol, and dicetyl phosphate, and their
mean vesicle sizes were adjusted to about 50 and 100 nm. (see,
e.g., Spuch and Navarro, Journal of Drug Delivery, vol. 2011,
Article ID 469679, 12 pages, 2011. doi:10.1155/2011/469679 for
review).
[0416] A liposome formulation may be mainly comprised of natural
phospholipids and lipids such as
1,2-distearoryl-sn-glycero-3-phosphatidyl choline (DSPC),
sphingomyelin, egg phosphatidylcholines and monosialoganglioside.
Since this formulation is made up of phospholipids only, liposomal
formulations have encountered many challenges, one of the ones
being the instability in plasma. Several attempts to overcome these
challenges have been made, specifically in the manipulation of the
lipid membrane. One of these attempts focused on the manipulation
of cholesterol. Addition of cholesterol to conventional
formulations reduces rapid release of the encapsulated bioactive
compound into the plasma or
1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE) increases the
stability (see, e.g., Spuch and Navarro, Journal of Drug Delivery,
vol. 2011, Article ID 469679, 12 pages, 2011.
doi:10.1155/2011/469679 for review).
[0417] In a particularly advantageous embodiment, Trojan Horse
liposomes (also known as Molecular Trojan Horses) are desirable and
protocols may be found at
http://cshprotocols.cshlp.org/content/2010/4/pdb.prot5407.long.
These particles allow delivery of a transgene to the entire brain
after an intravascular injection. Without being bound by
limitation, it is believed that neutral lipid particles with
specific antibodies conjugated to surface allow crossing of the
blood brain barrier via endocytosis. Applicant postulates utilizing
Trojan Horse Liposomes to deliver the CRISPR family of nucleases to
the brain via an intravascular injection, which would allow whole
brain transgenic animals without the need for embryonic
manipulation. About 1-5 g of DNA or RNA may be contemplated for in
vivo administration in liposomes.
[0418] In another embodiment, the CRISPR Cas system or components
thereof may be administered in liposomes, such as a stable
nucleic-acid-lipid particle (SNALP) (see, e.g., Morrissey et al.,
Nature Biotechnology, Vol. 23, No. 8, August 2005). Daily
intravenous injections of about 1, 3 or 5 mg/kg/day of a specific
CRISPR Cas9 targeted in a SNALP are contemplated. The daily
treatment may be over about three days and then weekly for about
five weeks. In another embodiment, a specific CRISPR Cas9
encapsulated SNALP) administered by intravenous injection to at
doses of about 1 or 2.5 mg/kg are also contemplated (see, e.g.,
Zimmerman et al., Nature Letters, Vol. 441, 4 May 2006). The SNALP
formulation may contain the lipids 3-N-[(wmethoxypoly(ethylene
glycol) 2000) carbamoyl]-1,2-dimyristyloxy-propylamine (PEG-C-DMA),
1,2-dilinoleyloxy-N,N-dimethyl-3-aminopropane (DLinDMA),
1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC) and cholesterol,
in a 2:40:10:48 molar percent ratio (see, e.g., Zimmerman et al.,
Nature Letters, Vol. 441, 4 May 2006).
[0419] In another embodiment, stable nucleic-acid-lipid particles
(SNALPs) have proven to be effective delivery molecules to highly
vascularized HepG2-derived liver tumors but not in poorly
vascularized HCT-116 derived liver tumors (see, e.g., Li, Gene
Therapy (2012) 19, 775-780). The SNALP liposomes may be prepared by
formulating D-Lin-DMA and PEG-C-DMA with
distearoylphosphatidylcholine (DSPC), Cholesterol and siRNA using a
25:1 lipid/siRNA ratio and a 48/40/10/2 molar ratio of
Cholesterol/D-Lin-DMA/DSPC/PEG-C-DMA. The resulted SNALP liposomes
are about 80-100 nm in size.
[0420] In yet another embodiment, a SNALP may comprise synthetic
cholesterol (Sigma-Aldrich, St Louis, Mo., USA),
dipalmitoylphosphatidylcholine (Avanti Polar Lipids, Alabaster,
Ala., USA), 3-N-[(w-methoxy poly(ethylene
glycol)2000)carbamoyl]-1,2-dimyrestyloxypropylamine, and cationic
1,2-dilinoleyloxy-3-N,Ndimethylaminopropane (see, e.g., Geisbert et
al., Lancet 2010; 375: 1896-905). A dosage of about 2 mg/kg total
CRISPR Cas9 per dose administered as, for example, a bolus
intravenous infusion may be contemplated.
[0421] In yet another embodiment, a SNALP may comprise synthetic
cholesterol (Sigma-Aldrich),
1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC; Avanti Polar
Lipids Inc.), PEG-cDMA, and
1,2-dilinoleyloxy-3-(N;N-dimethyl)aminopropane (DLinDMA) (see,
e.g., Judge, J. Clin. Invest. 119:661-673 (2009)). Formulations
used for in vivo studies may comprise a final lipid/RNA mass ratio
of about 9:1.
[0422] The safety profile of RNAi nanomedicines has been reviewed
by Barros and Gollob of Alnylam Pharmaceuticals (see, e.g.,
Advanced Drug Delivery Reviews 64 (2012) 1730-1737). The stable
nucleic acid lipid particle (SNALP) is comprised of four different
lipids--an ionizable lipid (DLinDMA) that is cationic at low pH, a
neutral helper lipid, cholesterol, and a diffusible polyethylene
glycol (PEG)-lipid. The particle is approximately 80 nm in diameter
and is charge-neutral at physiologic pH. During formulation, the
ionizable lipid serves to condense lipid with the anionic RNA
during particle formation. When positively charged under
increasingly acidic endosomal conditions, the ionizable lipid also
mediates the fusion of SNALP with the endosomal membrane enabling
release of RNA into the cytoplasm. The PEG-lipid stabilizes the
particle and reduces aggregation during formulation, and
subsequently provides a neutral hydrophilic exterior that improves
pharmacokinetic properties.
[0423] To date, two clinical programs have been initiated using
SNALP formulations with RNA. Tekmira Pharmaceuticals recently
completed a phase I single-dose study of SNALP-ApoB in adult
volunteers with elevated LDL cholesterol. ApoB is predominantly
expressed in the liver and jejunum and is essential for the
assembly and secretion of VLDL and LDL. Seventeen subjects received
a single dose of SNALP-ApoB (dose escalation across 7 dose levels).
There was no evidence of liver toxicity (anticipated as the
potential dose-limiting toxicity based on preclinical studies). One
(of two) subjects at the highest dose experienced flu-like symptoms
consistent with immune system stimulation, and the decision was
made to conclude the trial.
[0424] Alnylam Pharmaceuticals has similarly advanced ALN-TTR01,
which employs the SNALP technology described above and targets
hepatocyte production of both mutant and wild-type TTR to treat TTR
amyloidosis (ATTR). Three ATTR syndromes have been described:
familial amyloidotic polyneuropathy (FAP) and familial amyloidotic
cardiomyopathy (FAC) both caused by autosomal dominant mutations in
TTR; and senile systemic amyloidosis (SSA) cause by wildtype TTR. A
placebo-controlled, single dose-escalation phase I trial of
ALN-TTR01 was recently completed in patients with ATTR. ALN-TTR01
was administered as a 15-minute IV infusion to 31 patients (23 with
study drug and 8 with placebo) within a dose range of 0.01 to 1.0
mg/kg (based on siRNA). Treatment was well tolerated with no
significant increases in liver function tests. Infusion-related
reactions were noted in 3 of 23 patients at .gtoreq.0.4 mg/kg; all
responded to slowing of the infusion rate and all continued on
study. Minimal and transient elevations of serum cytokines IL-6,
IP-10 and IL-1ra were noted in two patients at the highest dose of
1 mg/kg (as anticipated from preclinical and NHP studies). Lowering
of serum TTR, the expected pharmacodynamics effect of ALN-TTR01,
was observed at 1 mg/kg.
[0425] In yet another embodiment, a SNALP may be made by
solubilizing a cationic lipid, DSPC, cholesterol and PEG-lipid
e.g., in ethanol, e.g., at a molar ratio of 40:10:40:10,
respectively (see, Semple et al., Nature Biotechnology, Volume 28
Number 2 Feb. 2010, pp. 172-177). The lipid mixture was added to an
aqueous buffer (50 mM citrate, pH 4) with mixing to a final ethanol
and lipid concentration of 30% (vol/vol) and 6.1 mg/ml,
respectively, and allowed to equilibrate at 22.degree. C. for 2 min
before extrusion. The hydrated lipids were extruded through two
stacked 80 nm pore-sized filters (Nuclepore) at 22.degree. C. using
a Lipex Extruder (Northern Lipids) until a vesicle diameter of
70-90 nm, as determined by dynamic light scattering analysis, was
obtained. This generally required 1-3 passes. The siRNA
(solubilized in a 50 mM citrate, pH 4 aqueous solution containing
30% ethanol) was added to the pre-equilibrated (35.degree. C.)
vesicles at a rate of .about.5 ml/min with mixing. After a final
target siRNA/lipid ratio of 0.06 (wt/wt) was reached, the mixture
was incubated for a further 30 min at 35.degree. C. to allow
vesicle reorganization and encapsulation of the siRNA. The ethanol
was then removed and the external buffer replaced with PBS (155 mM
NaCl, 3 mM Na.sub.2HPO.sub.4, 1 mM KH.sub.2PO.sub.4, pH 7.5) by
either dialysis or tangential flow diafiltration. siRNA were
encapsulated in SNALP using a controlled step-wise dilution method
process. The lipid constituents of KC2-SNALP were DLin-KC2-DMA
(cationic lipid), dipalmitoylphosphatidylcholine (DPPC; Avanti
Polar Lipids), synthetic cholesterol (Sigma) and PEG-C-DMA used at
a molar ratio of 57.1:7.1:34.3:1.4. Upon formation of the loaded
particles, SNALP were dialyzed against PBS and filter sterilized
through a 0.2 .mu.m filter before use. Mean particle sizes were
75-85 nm and 90-95% of the siRNA was encapsulated within the lipid
particles. The final siRNA/lipid ratio in formulations used for in
vivo testing was .about.0.15 (wt/wt). LNP-siRNA systems containing
Factor VII siRNA were diluted to the appropriate concentrations in
sterile PBS immediately before use and the formulations were
administered intravenously through the lateral tail vein in a total
volume of 10 ml/kg. This method and these delivery systems may be
extrapolated to the CRISPR Cas9 system of the present
invention.
Other Lipids
[0426] Other cationic lipids, such as amino lipid
2,2-dilinoleyl-4-dimethylaminoethyl-[1,3]-dioxolane (DLin-KC2-DMA)
may be utilized to encapsulate CRISPR Cas9 or components thereof or
nucleic acid molecule(s) coding therefor e.g., similar to SiRNA
(see, e.g., Jayaraman, Angew. Chem. Int. Ed. 2012, 51, 8529-8533),
and hence may be employed in the practice of the invention. A
preformed vesicle with the following lipid composition may be
contemplated: amino lipid, distearoylphosphatidylcholine (DSPC),
cholesterol and (R)-2,3-bis(octadecyloxy) propyl-1-(methoxy
poly(ethylene glycol)2000)propylcarbamate (PEG-lipid) in the molar
ratio 40/10/40/10, respectively, and a FVII siRNA/total lipid ratio
of approximately 0.05 (w/w). To ensure a narrow particle size
distribution in the range of 70-90 nm and a low polydispersity
index of 0.11.+-.0.04 (n=56), the particles may be extruded up to
three times through 80 nm membranes prior to adding the guide RNA.
Particles containing the highly potent amino lipid 16 may be used,
in which the molar ratio of the four lipid components 16, DSPC,
cholesterol and PEG-lipid (50/10/38.5/1.5) which may be further
optimized to enhance in vivo activity.
[0427] Michael S D Kormann et al. ("Expression of therapeutic
proteins after delivery of chemically modified mRNA in mice: Nature
Biotechnology, Volume: 29, Pages: 154-157 (2011)) describes the use
of lipid envelopes to deliver RNA. Use of lipid envelopes is also
preferred in the present invention.
[0428] In another embodiment, lipids may be formulated with the
CRISPR Cas system of the present invention or component(s) thereof
or nucleic acid molecule(s) coding therefor to form lipid
nanoparticles (LNPs). Lipids include, but are not limited to,
DLin-KC2-DMA4, C12-200 and colipids disteroylphosphatidyl choline,
cholesterol, and PEG-DMG may be formulated with CRISPR Cas instead
of siRNA (see, e.g., Novobrantseva, Molecular Therapy--Nucleic
Acids (2012) 1, e4; doi:10.1038/mtna.2011.3) using a spontaneous
vesicle formation procedure. The component molar ratio may be about
50/10/38.5/1.5 (DLin-KC2-DMA or C12-200/disteroylphosphatidyl
choline/cholesterol/PEG-DMG). The final lipid:siRNA weight ratio
may be .about.12:1 and 9:1 in the case of DLin-KC2-DMA and C12-200
lipid nanoparticles (LNPs), respectively. The formulations may have
mean particle diameters of .about.80 nm with >90% entrapment
efficiency. A 3 mg/kg dose may be contemplated.
[0429] Tekmira has a portfolio of approximately 95 patent families,
in the U.S. and abroad, that are directed to various aspects of
LNPs and LNP formulations (see, e.g., U.S. Pat. Nos. 7,982,027;
7,799,565; 8,058,069; 8,283,333; 7,901,708; 7,745,651; 7,803,397;
8,101,741; 8,188,263; 7,915,399; 8,236,943 and 7,838,658 and
European Pat. Nos 1766035; 1519714; 1781593 and 1664316), all of
which may be used and/or adapted to the present invention.
[0430] The CRISPR Cas system or components thereof or nucleic acid
molecule(s) coding therefor may be delivered encapsulated in PLGA
Microspheres such as that further described in US published
applications 20130252281 and 20130245107 and 20130244279 (assigned
to Moderna Therapeutics) which relate to aspects of formulation of
compositions comprising modified nucleic acid molecules which may
encode a protein, a protein precursor, or a partially or fully
processed form of the protein or a protein precursor. The
formulation may have a molar ratio 50:10:38.5:1.5-3.0 (cationic
lipid:fusogenic lipid:cholesterol:PEG lipid). The PEG lipid may be
selected from, but is not limited to PEG-c-DOMG, PEG-DMG. The
fusogenic lipid may be DSPC. See also, Schrum et al., Delivery and
Formulation of Engineered Nucleic Acids, US published application
20120251618.
[0431] Nanomerics' technology addresses bioavailability challenges
for a broad range of therapeutics, including low molecular weight
hydrophobic drugs, peptides, and nucleic acid based therapeutics
(plasmid, siRNA, miRNA). Specific administration routes for which
the technology has demonstrated clear advantages include the oral
route, transport across the blood-brain-barrier, delivery to solid
tumors, as well as to the eye. See, e.g., Mazza et al., 2013, ACS
Nano. 2013 Feb. 26; 7(2):1016-26; Uchegbu and Siew, 2013, J Pharm
Sci. 102(2):305-10 and Lalatsa et al., 2012, J Control Release.
2012 Jul. 20; 161(2):523-36.
[0432] US Patent Publication No. 20050019923 describes cationic
dendrimers for delivering bioactive molecules, such as
polynucleotide molecules, peptides and polypeptides and/or
pharmaceutical agents, to a mammalian body. The dendrimers are
suitable for targeting the delivery of the bioactive molecules to,
for example, the liver, spleen, lung, kidney or heart (or even the
brain). Dendrimers are synthetic 3-dimensional macromolecules that
are prepared in a step-wise fashion from simple branched monomer
units, the nature and functionality of which can be easily
controlled and varied. Dendrimers are synthesized from the repeated
addition of building blocks to a multifunctional core (divergent
approach to synthesis), or towards a multifunctional core
(convergent approach to synthesis) and each addition of a
3-dimensional shell of building blocks leads to the formation of a
higher generation of the dendrimers. Polypropylenimine dendrimers
start from a diaminobutane core to which is added twice the number
of amino groups by a double Michael addition of acrylonitrile to
the primary amines followed by the hydrogenation of the nitriles.
This results in a doubling of the amino groups. Polypropylenimine
dendrimers contain 100% protonable nitrogens and up to 64 terminal
amino groups (generation 5, DAB 64). Protonable groups are usually
amine groups which are able to accept protons at neutral pH. The
use of dendrimers as gene delivery agents has largely focused on
the use of the polyamidoamine. and phosphorous containing compounds
with a mixture of amine/amide or N--P(O.sub.2)S as the conjugating
units respectively with no work being reported on the use of the
lower generation polypropylenimine dendrimers for gene delivery.
Polypropylenimine dendrimers have also been studied as pH sensitive
controlled release systems for drug delivery and for their
encapsulation of guest molecules when chemically modified by
peripheral amino acid groups. The cytotoxicity and interaction of
polypropylenimine dendrimers with DNA as well as the transfection
efficacy of DAB 64 has also been studied.
[0433] US Patent Publication No. 20050019923 is based upon the
observation that, contrary to earlier reports, cationic dendrimers,
such as polypropylenimine dendrimers, display suitable properties,
such as specific targeting and low toxicity, for use in the
targeted delivery of bioactive molecules, such as genetic material.
In addition, derivatives of the cationic dendrimer also display
suitable properties for the targeted delivery of bioactive
molecules. See also, Bioactive Polymers, US published application
20080267903, which discloses "Various polymers, including cationic
polyamine polymers and dendrimeric polymers, are shown to possess
anti-proliferative activity, and may therefore be useful for
treatment of disorders characterised by undesirable cellular
proliferation such as neoplasms and tumors, inflammatory disorders
(including autoimmune disorders), psoriasis and atherosclerosis.
The polymers may be used alone as active agents, or as delivery
vehicles for other therapeutic agents, such as drug molecules or
nucleic acids for gene therapy. In such cases, the polymers' own
intrinsic anti-tumor activity may complement the activity of the
agent to be delivered." The disclosures of these patent
publications may be employed in conjunction with herein teachings
for delivery of CRISPR Cas system(s) or component(s) thereof or
nucleic acid molecule(s) coding therefor.
Supercharged Proteins
[0434] Supercharged proteins are a class of engineered or naturally
occurring proteins with unusually high positive or negative net
theoretical charge and may be employed in delivery of CRISPR Cas9
system(s) or component(s) thereof or nucleic acid molecule(s)
coding therefor. Both supernegatively and superpositively charged
proteins exhibit a remarkable ability to withstand thermally or
chemically induced aggregation. Superpositively charged proteins
are also able to penetrate mammalian cells. Associating cargo with
these proteins, such as plasmid DNA, RNA, or other proteins, can
enable the functional delivery of these macromolecules into
mammalian cells both in vitro and in vivo. David Liu's lab reported
the creation and characterization of supercharged proteins in 2007
(Lawrence et al., 2007, Journal of the American Chemical Society
129, 10110-10112).
[0435] The nonviral delivery of RNA and plasmid DNA into mammalian
cells are valuable both for research and therapeutic applications
(Akinc et al., 2010, Nat. Biotech. 26, 561-569). Purified +36 GFP
protein (or other superpositively charged protein) is mixed with
RNAs in the appropriate serum-free media and allowed to complex
prior addition to cells. Inclusion of serum at this stage inhibits
formation of the supercharged protein-RNA complexes and reduces the
effectiveness of the treatment. The following protocol has been
found to be effective for a variety of cell lines (McNaughton et
al., 2009, Proc. Natl. Acad. Sci. USA 106, 6111-6116). However,
pilot experiments varying the dose of protein and RNA should be
performed to optimize the procedure for specific cell lines.
[0436] (1) One day before treatment, plate 1.times.10.sup.5 cells
per well in a 48-well plate.
[0437] (2) On the day of treatment, dilute purified +36 GFP protein
in serumfree media to a final concentration 200 nM. Add RNA to a
final concentration of 50 nM. Vortex to mix and incubate at room
temperature for 10 min.
[0438] (3) During incubation, aspirate media from cells and wash
once with PBS.
[0439] (4) Following incubation of +36 GFP and RNA, add the
protein-RNA complexes to cells.
[0440] (5) Incubate cells with complexes at 37.degree. C. for 4
h.
[0441] (6) Following incubation, aspirate the media and wash three
times with 20 U/mL heparin PBS. Incubate cells with
serum-containing media for a further 48 h or longer depending upon
the assay for activity.
[0442] (7) Analyze cells by immunoblot, qPCR, phenotypic assay, or
other appropriate method.
[0443] David Liu's lab has further found +36 GFP to be an effective
plasmid delivery reagent in a range of cells. As plasmid DNA is a
larger cargo than siRNA, proportionately more +36 GFP protein is
required to effectively complex plasmids. For effective plasmid
delivery Applicants have developed a variant of +36 GFP bearing a
C-terminal HA2 peptide tag, a known endosome-disrupting peptide
derived from the influenza virus hemagglutinin protein. The
following protocol has been effective in a variety of cells, but as
above it is advised that plasmid DNA and supercharged protein doses
be optimized for specific cell lines and delivery applications.
[0444] (1) One day before treatment, plate 1.times.10.sup.5 per
well in a 48-well plate.
[0445] (2) On the day of treatment, dilute purified 36 GFP protein
in serumfree media to a final concentration 2 mM. Add 1 mg of
plasmid DNA. Vortex to mix and incubate at room temperature for 10
min.
[0446] (3) During incubation, aspirate media from cells and wash
once with PBS.
[0447] (4) Following incubation of 36 GFP and plasmid DNA, gently
add the protein-DNA complexes to cells.
[0448] (5) Incubate cells with complexes at 37 C for 4 h.
[0449] (6) Following incubation, aspirate the media and wash with
PBS. Incubate cells in serum-containing media and incubate for a
further 24-48 h.
[0450] (7) Analyze plasmid delivery (e.g., by plasmid-driven gene
expression) as appropriate.
[0451] See also, e.g., McNaughton et al., Proc. Natl. Acad. Sci.
USA 106, 6111-6116 (2009); Cronican et al., ACS Chemical Biology 5,
747-752 (2010); Cronican et al., Chemistry & Biology 18,
833-838 (2011); Thompson et al., Methods in Enzymology 503, 293-319
(2012); Thompson, D. B., et al., Chemistry & Biology 19 (7),
831-843 (2012). The methods of the super charged proteins may be
used and/or adapted for delivery of the CRISPR Cas system of the
present invention. These systems of Dr. Lui and documents herein in
inconjunction with herein teachints can be employed in the delivery
of CRISPR Cas system(s) or component(s) thereof or nucleic acid
molecule(s) coding therefor.
Cell Penetrating Peptides (CPPs)
[0452] In yet another embodiment, cell penetrating peptides (CPPs)
are contemplated for the delivery of the CRISPR Cas system. CPPs
are short peptides that facilitate cellular uptake of various
molecular cargo (from nanosize particles to small chemical
molecules and large fragments of DNA). The term "cargo" as used
herein includes but is not limited to the group consisting of
therapeutic agents, diagnostic probes, peptides, nucleic acids,
antisense oligonucleotides, plasmids, proteins, particles including
nanoparticles, liposomes, chromophores, small molecules and
radioactive materials. In aspects of the invention, the cargo may
also comprise any component of the CRISPR Cas system or the entire
functional CRISPR Cas system. Aspects of the present invention
further provide methods for delivering a desired cargo into a
subject comprising: (a) preparing a complex comprising the cell
penetrating peptide of the present invention and a desired cargo,
and (b) orally, intraarticularly, intraperitoneally, intrathecally,
intrarterially, intranasally, intraparenchymally, subcutaneously,
intramuscularly, intravenously, dermally, intrarectally, or
topically administering the complex to a subject. The cargo is
associated with the peptides either through chemical linkage via
covalent bonds or through non-covalent interactions.
[0453] The function of the CPPs are to deliver the cargo into
cells, a process that commonly occurs through endocytosis with the
cargo delivered to the endosomes of living mammalian cells.
Cell-penetrating peptides are of different sizes, amino acid
sequences, and charges but all CPPs have one distinct
characteristic, which is the ability to translocate the plasma
membrane and facilitate the delivery of various molecular cargoes
to the cytoplasm or an organelle. CPP translocation may be
classified into three main entry mechanisms: direct penetration in
the membrane, endocytosis-mediated entry, and translocation through
the formation of a transitory structure. CPPs have found numerous
applications in medicine as drug delivery agents in the treatment
of different diseases including cancer and virus inhibitors, as
well as contrast agents for cell labeling. Examples of the latter
include acting as a carrier for GFP, Mill contrast agents, or
quantum dots. CPPs hold great potential as in vitro and in vivo
delivery vectors for use in research and medicine. CPPs typically
have an amino acid composition that either contains a high relative
abundance of positively charged amino acids such as lysine or
arginine or has sequences that contain an alternating pattern of
polar/charged amino acids and non-polar, hydrophobic amino acids.
These two types of structures are referred to as polycationic or
amphipathic, respectively. A third class of CPPs are the
hydrophobic peptides, containing only apolar residues, with low net
charge or have hydrophobic amino acid groups that are crucial for
cellular uptake. One of the initial CPPs discovered was the
trans-activating transcriptional activator (Tat) from Human
Immunodeficiency Virus 1 (HIV-1) which was found to be efficiently
taken up from the surrounding media by numerous cell types in
culture. Since then, the number of known CPPs has expanded
considerably and small molecule synthetic analogues with more
effective protein transduction properties have been generated. CPPs
include but are not limited to Penetratin, Tat (48-60),
Transportan, and (R-AhX-R)4) (SEQ ID NO: 44)
(Ahx=aminohexanoyl).
[0454] U.S. Pat. No. 8,372,951, provides a CPP derived from
eosinophil cationic protein (ECP) which exhibits highly
cell-penetrating efficiency and low toxicity. Aspects of delivering
the CPP with its cargo into a vertebrate subject are also provided.
Further aspects of CPPs and their delivery are described in U.S.
Pat. Nos. 8,575,305; 8,614,194 and 8,044,019. CPPs can be used to
deliver the CRISPR-Cas system or components thereof. That CPPs can
be employed to deliver the CRISPR-Cas system or components thereof
is also provided in the manuscript "Gene disruption by
cell-penetrating peptide-mediated delivery of Cas9 protein and
guide RNA", by Suresh Ramakrishna, Abu-Bonsrah Kwaku Dad, Jagadish
Beloor, et al. Genome Res. 2014 Apr. 2. [Epub ahead of print],
incorporated by reference in its entirety, wherein it is
demonstrated that treatment with CPP-conjugated recombinant Cas9
protein and CPP-complexed guide RNAs lead to endogenous gene
disruptions in human cell lines. In the paper the Cas9 protein was
conjugated to CPP via a thioether bond, whereas the guide RNA was
complexed with CPP, forming condensed, positively charged
particles. It was shown that simultaneous and sequential treatment
of human cells, including embryonic stem cells, dermal fibroblasts,
HEK293T cells, HeLa cells, and embryonic carcinoma cells, with the
modified Cas9 and guide RNA led to efficient gene disruptions with
reduced off-target mutations relative to plasmid transfections.
Implantable Devices
[0455] In another embodiment, implantable devices are also
contemplated for delivery of the CRISPR Cas system or component(s)
thereof or nucleic acid molecule(s) coding therefor. For example,
US Patent Publication 20110195123 discloses an implantable medical
device which elutes a drug locally and in prolonged period is
provided, including several types of such a device, the treatment
modes of implementation and methods of implantation. The device
comprising of polymeric substrate, such as a matrix for example,
that is used as the device body, and drugs, and in some cases
additional scaffolding materials, such as metals or additional
polymers, and materials to enhance visibility and imaging. An
implantable delivery device can be advantageous in providing
release locally and over a prolonged period, where drug is released
directly to the extracellular matrix (ECM) of the diseased area
such as tumor, inflammation, degeneration or for symptomatic
objectives, or to injured smooth muscle cells, or for prevention.
One kind of drug is RNA, as disclosed above, and this system may be
used/and or adapted to the CRISPR Cas system of the present
invention. The modes of implantation in some embodiments are
existing implantation procedures that are developed and used today
for other treatments, including brachytherapy and needle biopsy. In
such cases the dimensions of the new implant described in this
invention are similar to the original implant. Typically a few
devices are implanted during the same treatment procedure.
[0456] US Patent Publication 20110195123 provides a drug delivery
implantable or insertable system, including systems applicable to a
cavity such as the abdominal cavity and/or any other type of
administration in which the drug delivery system is not anchored or
attached, comprising a biostable and/or degradable and/or
bioabsorbable polymeric substrate, which may for example optionally
be a matrix. It should be noted that the term "insertion" also
includes implantation. The drug delivery system is preferably
implemented as a "Loder" as described in US Patent Publication
20110195123.
[0457] The polymer or plurality of polymers are biocompatible,
incorporating an agent and/or plurality of agents, enabling the
release of agent at a controlled rate, wherein the total volume of
the polymeric substrate, such as a matrix for example, in some
embodiments is optionally and preferably no greater than a maximum
volume that permits a therapeutic level of the agent to be reached.
As a non-limiting example, such a volume is preferably within the
range of 0.1 m.sup.3 to 1000 mm.sup.3, as required by the volume
for the agent load. The Loder may optionally be larger, for example
when incorporated with a device whose size is determined by
functionality, for example and without limitation, a knee joint, an
intra-uterine or cervical ring and the like.
[0458] The drug delivery system (for delivering the composition) is
designed in some embodiments to preferably employ degradable
polymers, wherein the main release mechanism is bulk erosion; or in
some embodiments, non degradable, or slowly degraded polymers are
used, wherein the main release mechanism is diffusion rather than
bulk erosion, so that the outer part functions as membrane, and its
internal part functions as a drug reservoir, which practically is
not affected by the surroundings for an extended period (for
example from about a week to about a few months). Combinations of
different polymers with different release mechanisms may also
optionally be used. The concentration gradient at the surface is
preferably maintained effectively constant during a significant
period of the total drug releasing period, and therefore the
diffusion rate is effectively constant (termed "zero mode"
diffusion). By the term "constant" it is meant a diffusion rate
that is preferably maintained above the lower threshold of
therapeutic effectiveness, but which may still optionally feature
an initial burst and/or may fluctuate, for example increasing and
decreasing to a certain degree. The diffusion rate is preferably so
maintained for a prolonged period, and it can be considered
constant to a certain level to optimize the therapeutically
effective period, for example the effective silencing period.
[0459] The drug delivery system optionally and preferably is
designed to shield the nucleotide based therapeutic agent from
degradation, whether chemical in nature or due to attack from
enzymes and other factors in the body of the subject.
[0460] The drug delivery system as described in US Patent
Publication 20110195123 is optionally associated with sensing
and/or activation appliances that are operated at and/or after
implantation of the device, by non and/or minimally invasive
methods of activation and/or acceleration/deceleration, for example
optionally including but not limited to thermal heating and
cooling, laser beams, and ultrasonic, including focused ultrasound
and/or RF (radiofrequency) methods or devices.
[0461] According to some embodiments of US Patent Publication
20110195123, the site for local delivery may optionally include
target sites characterized by high abnormal proliferation of cells,
and suppressed apoptosis, including tumors, active and or chronic
inflammation and infection including autoimmune diseases states,
degenerating tissue including muscle and nervous tissue, chronic
pain, degenerative sites, and location of bone fractures and other
wound locations for enhancement of regeneration of tissue, and
injured cardiac, smooth and striated muscle.
[0462] The site for implantation of the composition, or target
site, preferably features a radius, area and/or volume that is
sufficiently small for targeted local delivery. For example, the
target site optionally has a diameter in a range of from about 0.1
mm to about 5 cm.
[0463] The location of the target site is preferably selected for
maximum therapeutic efficacy. For example, the composition of the
drug delivery system (optionally with a device for implantation as
described above) is optionally and preferably implanted within or
in the proximity of a tumor environment, or the blood supply
associated thereof.
[0464] For example the composition (optionally with the device) is
optionally implanted within or in the proximity to pancreas,
prostate, breast, liver, via the nipple, within the vascular system
and so forth.
[0465] The target location is optionally selected from the group
comprising, consisting essentially of, or consisting of (as
non-limiting examples only, as optionally any site within the body
may be suitable for implanting a Loder): 1. brain at degenerative
sites like in Parkinson or Alzheimer disease at the basal ganglia,
white and gray matter; 2. spine as in the case of amyotrophic
lateral sclerosis (ALS); 3. uterine cervix to prevent HPV
infection; 4. active and chronic inflammatory joints; 5. dermis as
in the case of psoriasis; 6. sympathetic and sensoric nervous sites
for analgesic effect; 7. Intra osseous implantation; 8. acute and
chronic infection sites; 9. Intra vaginal; 10. Inner ear-auditory
system, labyrinth of the inner ear, vestibular system; 11. Intra
tracheal; 12. Intra-cardiac; coronary, epicardiac; 13. urinary
bladder; 14. biliary system; 15. parenchymal tissue including and
not limited to the kidney, liver, spleen; 16. lymph nodes; 17.
salivary glands; 18. dental gums; 19. Intra-articular (into
joints); 20. Intra-ocular; 21. Brain tissue; 22. Brain ventricles;
23. Cavities, including abdominal cavity (for example but without
limitation, for ovary cancer); 24. Intra esophageal and 25. Intra
rectal.
[0466] Optionally insertion of the system (for example a device
containing the composition) is associated with injection of
material to the ECM at the target site and the vicinity of that
site to affect local pH and/or temperature and/or other biological
factors affecting the diffusion of the drug and/or drug kinetics in
the ECM, of the target site and the vicinity of such a site.
[0467] Optionally, according to some embodiments, the release of
said agent could be associated with sensing and/or activation
appliances that are operated prior and/or at and/or after
insertion, by non and/or minimally invasive and/or else methods of
activation and/or acceleration/deceleration, including laser beam,
radiation, thermal heating and cooling, and ultrasonic, including
focused ultrasound and/or RF (radiofrequency) methods or devices,
and chemical activators.
[0468] According to other embodiments of US Patent Publication
20110195123, the drug preferably comprises a RNA, for example for
localized cancer cases in breast, pancreas, brain, kidney, bladder,
lung, and prostate as described below. Although exemplified with
RNAi, many drugs are applicable to be encapsulated in Loder, and
can be used in association with this invention, as long as such
drugs can be encapsulated with the Loder substrate, such as a
matrix for example, and this system may be used and/or adapted to
deliver the CRISPR Cas system of the present invention.
[0469] As another example of a specific application, neuro and
muscular degenerative diseases develop due to abnormal gene
expression. Local delivery of RNAs may have therapeutic properties
for interfering with such abnormal gene expression. Local delivery
of anti apoptotic, anti inflammatory and anti degenerative drugs
including small drugs and macromolecules may also optionally be
therapeutic. In such cases the Loder is applied for prolonged
release at constant rate and/or through a dedicated device that is
implanted separately. All of this may be used and/or adapted to the
CRISPR Cas system of the present invention.
[0470] As yet another example of a specific application,
psychiatric and cognitive disorders are treated with gene
modifiers. Gene knockdown is a treatment option. Loders locally
delivering agents to central nervous system sites are therapeutic
options for psychiatric and cognitive disorders including but not
limited to psychosis, bi-polar diseases, neurotic disorders and
behavioral maladies. The Loders could also deliver locally drugs
including small drugs and macromolecules upon implantation at
specific brain sites. All of this may be used and/or adapted to the
CRISPR Cas system of the present invention.
[0471] As another example of a specific application, silencing of
innate and/or adaptive immune mediators at local sites enables the
prevention of organ transplant rejection. Local delivery of RNAs
and immunomodulating reagents with the Loder implanted into the
transplanted organ and/or the implanted site renders local immune
suppression by repelling immune cells such as CD8 activated against
the transplanted organ. All of this may be used/and or adapted to
the CRISPR Cas system of the present invention.
[0472] As another example of a specific application, vascular
growth factors including VEGFs and angiogenin and others are
essential for neovascularization. Local delivery of the factors,
peptides, peptidomimetics, or suppressing their repressors is an
important therapeutic modality; silencing the repressors and local
delivery of the factors, peptides, macromolecules and small drugs
stimulating angiogenesis with the Loder is therapeutic for
peripheral, systemic and cardiac vascular disease.
[0473] The method of insertion, such as implantation, may
optionally already be used for other types of tissue implantation
and/or for insertions and/or for sampling tissues, optionally
without modifications, or alternatively optionally only with
non-major modifications in such methods. Such methods optionally
include but are not limited to brachytherapy methods, biopsy,
endoscopy with and/or without ultrasound, such as ERCP,
stereotactic methods into the brain tissue, Laparoscopy, including
implantation with a laparoscope into joints, abdominal organs, the
bladder wall and body cavity.
[0474] Implantable device technology herein discussed can be
employed with herein teachings and hence by this disclosure and the
knowledge in the art, CRISPR-Cas system or components thereof or
nucleic acid molecules thereof or encoding or providing components
may be delivered via an implantable device.
Patient-Specific Screening Methods
[0475] A CRISPR-Cas system that targets nucleotide, e.g.,
trinucleotide repeats can be used to screen patients or patent
samples for the presence of such repeats. The repeats can be the
target of the RNA of the CRISPR-Cas system, and if there is binding
thereto by the CRISPR-Cas system, that binding can be detected, to
thereby indicate that such a repeat is present. Thus, a CRISPR-Cas
system can be used to screen patients or patient samples for the
presence of the repeat. The patient can then be administered
suitable compound(s) to address the condition; or, can be
administed a CRISPR-Cas system to bind to and cause insertion,
deletion or mutation and alleviate the condition.
CRISPR Effector Protein mRNA and Guide RNA
[0476] CRISPR enzyme mRNA and guide RNA might also be delivered
separately. CRISPR enzyme mRNA can be delivered prior to the guide
RNA to give time for CRISPR enzyme to be expressed. CRISPR enzyme
mRNA might be administered 1-12 hours (preferably around 2-6 hours)
prior to the administration of guide RNA.
[0477] Alternatively, CRISPR enzyme mRNA and guide RNA can be
administered together. Advantageously, a second booster dose of
guide RNA can be administered 1-12 hours (preferably around 2-6
hours) after the initial administration of CRISPR enzyme mRNA+guide
RNA.
[0478] The CRISPR effector protein of the present invention, i.e. a
Cas9 effector protein is sometimes referred to herein as a CRISPR
Enzyme. It will be appreciated that the effector protein is based
on or derived from an enzyme, so the term `effector protein`
certainly includes `enzyme` in some embodiments. However, it will
also be appreciated that the effector protein may, as required in
some embodiments, have DNA or RNA binding, but not necessarily
cutting or nicking, activity, including a dead-Cas effector protein
function.
[0479] Additional administrations of CRISPR enzyme mRNA and/or
guide RNA might be useful to achieve the most efficient levels of
genome modification. In some embodiments, phenotypic alteration is
preferably the result of genome modification when a genetic disease
is targeted, especially in methods of therapy and preferably where
a repair template is provided to correct or alter the
phenotype.
[0480] In some embodiments diseases that may be targeted include
those concerned with disease-causing splice defects.
[0481] In some embodiments, cellular targets include Hemopoietic
Stem/Progenitor Cells (CD34+); Human T cells; and Eye (retinal
cells)--for example photoreceptor precursor cells.
[0482] In some embodiments Gene targets include: Human Beta
Globin--HBB (for treating Sickle Cell Anemia, including by
stimulating gene-conversion (using closely related HBD gene as an
endogenous template)); CD3 (T-Cells); and CEP920--retina (eye).
[0483] In some embodiments disease targets also include: cancer;
Sickle Cell Anemia (based on a point mutation); HIV;
Beta-Thalassemia; and ophthalmic or ocular disease--for example
Leber Congenital Amaurosis (LCA)-causing Splice Defect.
[0484] In some embodiments delivery methods include: Cationic Lipid
Mediated "direct" delivery of Enzyme-Guide complex
(RiboNucleoProtein) and electroporation of plasmid DNA.
[0485] Inventive methods can further comprise delivery of
templates, such as repair templates, which may be dsODN or ssODN,
see below. Delivery of templates may be via the cotemporaneous or
separate from delivery of any or all the CRISPR enzyme, guide,
tracr mate or tracrRNA and via the same delivery mechanism or
different. In some embodiments, it is preferred that the template
is delivered together with the guide, tracr mate and/or tracrRNA
and, preferably, also the CRISPR enzyme. An example may be an AAV
vector where the CRISPR enzyme is SaCas9 (with the N580
mutation).
[0486] Inventive methods can further comprise: (a) delivering to
the cell a double-stranded oligodeoxynucleotide (dsODN) comprising
overhangs complimentary to the overhangs created by said double
strand break, wherein said dsODN is integrated into the locus of
interest; or--(b) delivering to the cell a single-stranded
oligodeoxynucleotide (ssODN), wherein said ssODN acts as a template
for homology directed repair of said double strand break. Inventive
methods can be for the prevention or treatment of disease in an
individual, optionally wherein said disease is caused by a defect
in said locus of interest. Inventive methods can be conducted in
vivo in the individual or ex vivo on a cell taken from the
individual, optionally wherein said cell is returned to the
individual.
[0487] For minimization of toxicity and off-target effect, it will
be important to control the concentration of CRISPR enzyme mRNA and
guide RNA delivered. Optimal concentrations of CRISPR enzyme mRNA
and guide RNA can be determined by testing different concentrations
in a cellular or animal model and using deep sequencing the analyze
the extent of modification at potential off-target genomic loci.
For example, for the guide sequence targeting
5'-GAGTCCGAGCAGAAGAAGAA-3' (SEQ ID NO: 45) in the EMX1 gene of the
human genome, deep sequencing can be used to assess the level of
modification at the following two off-target loci, 1:
5'-GAGTCCTAGCAGGAGAAGAA-3' (SEQ ID NO: 46) and 2:
5'-GAGTCTAAGCAGAAGAAGAA-3' (SEQ ID NO: 47). The concentration that
gives the highest level of on-target modification while minimizing
the level of off-target modification should be chosen for in vivo
delivery.
Inducible Systems
[0488] In some embodiments, a CRISPR enzyme may form a component of
an inducible system. The inducible nature of the system would allow
for spatiotemporal control of gene editing or gene expression using
a form of energy. The form of energy may include but is not limited
to electromagnetic radiation, sound energy, chemical energy and
thermal energy. Examples of inducible system include tetracycline
inducible promoters (Tet-On or Tet-Off), small molecule two-hybrid
transcription activations systems (FKBP, ABA, etc), or light
inducible systems (Phytochrome, LOV domains, or cryptochrome). In
one embodiment, the CRISPR enzyme may be a part of a Light
Inducible Transcriptional Effector (LITE) to direct changes in
transcriptional activity in a sequence-specific manner. The
components of a LITE may include a CRISPR enzyme, a
light-responsive cytochrome heterodimer (e.g. from Arabidopsis
thaliana), and a transcriptional activation/repression domain.
Further examples of inducible DNA binding proteins and methods for
their use are provided in U.S. 61/736,465, U.S. 61/721,283 and WO
2014/018423, which is hereby incorporated by reference in its
entirety.
Self-Inactivating Systems
[0489] Once all copies of a gene in the genome of a cell have been
edited, continued CRISRP/Cas9 expression in that cell is no longer
necessary. Indeed, sustained expression would be undesirable in
case of off-target effects at unintended genomic sites, etc. Thus
time-limited expression would be useful. Inducible expression
offers one approach, but in addition Applicants have engineered a
Self-Inactivating CRISPR-Cas9 system that relies on the use of a
non-coding guide target sequence within the CRISPR vector itself.
Thus, after expression begins, the CRISPR system will lead to its
own destruction, but before destruction is complete it will have
time to edit the genomic copies of the target gene (which, with a
normal point mutation in a diploid cell, requires at most two
edits). Simply, the self inactivating CRISPR-Cas system includes
additional RNA (i.e., guide RNA) that targets the coding sequence
for the CRISPR enzyme itself or that targets one or more non-coding
guide target sequences complementary to unique sequences present in
one or more of the following:
(a) within the promoter driving expression of the non-coding RNA
elements, (b) within the promoter driving expression of the Cas9
gene, (c) within 100 bp of the ATG translational start codon in the
Cas9 coding sequence, (d) within the inverted terminal repeat (iTR)
of a viral delivery vector, e.g., in the AAV genome.
[0490] Furthermore, that RNA can be delivered via a vector, e.g., a
separate vector or the same vector that is encoding the CRISPR
complex. When provided by a separate vector, the CRISPR RNA that
targets Cas expression can be administered sequentially or
simultaneously. When administered sequentially, the CRISPR RNA that
targets Cas expression is to be delivered after the CRISPR RNA that
is intended for e.g. gene editing or gene engineering. This period
may be a period of minutes (e.g. 5 minutes, 10 minutes, 20 minutes,
30 minutes, 45 minutes, 60 minutes). This period may be a period of
hours (e.g. 2 hours, 4 hours, 6 hours, 8 hours, 12 hours, 24
hours). This period may be a period of days (e.g. 2 days, 3 days, 4
days, 7 days). This period may be a period of weeks (e.g. 2 weeks,
3 weeks, 4 weeks). This period may be a period of months (e.g. 2
months, 4 months, 8 months, 12 months). This period may be a period
of years (2 years, 3 years, 4 years). In this fashion, the Cas
enzyme associates with a first gRNA/chiRNA capable of hybridizing
to a first target, such as a genomic locus or loci of interest and
undertakes the function(s) desired of the CRISPR-Cas system (e.g.,
gene engineering); and subsequently the Cas9 enzyme may then
associate with the second gRNA/chiRNA capable of hybridizing to the
sequence comprising at least part of the Cas9 or CRISPR cassette.
Where the gRNA/chiRNA targets the sequences encoding expression of
the Cas9 protein, the enzyme becomes impeded and the system becomes
self inactivating. In the same manner, CRISPR RNA that targets Cas9
expression applied via, for example liposome, lipofection,
particles, microvesicles as explained herein, may be administered
sequentially or simultaneously. Similarly, self-inactivation may be
used for inactivation of one or more guide RNA used to target one
or more targets.
[0491] In some aspects, a single gRNA is provided that is capable
of hybridization to a sequence downstream of a CRISPR enzyme start
codon, whereby after a period of time there is a loss of the CRISPR
enzyme expression. In some aspects, one or more gRNA(s) are
provided that are capable of hybridization to one or more coding or
non-coding regions of the polynucleotide encoding the CRISPR-Cas
system, whereby after a period of time there is a inactivation of
one or more, or in some cases all, of the CRISPR-Cas system. In
some aspects of the system, and not to be limited by theory, the
cell may comprise a plurality of CRISPR-Cas complexes, wherein a
first subset of CRISPR complexes comprise a first chiRNA capable of
targeting a genomic locus or loci to be edited, and a second subset
of CRISPR complexes comprise at least one second chiRNA capable of
targeting the polynucleotide encoding the CRISPR-Cas system,
wherein the first subset of CRISPR-Cas complexes mediate editing of
the targeted genomic locus or loci and the second subset of CRISPR
complexes eventually inactivate the CRISPR-Cas system, thereby
inactivating further CRISPR-Cas expression in the cell.
[0492] Thus the invention provides a CRISPR-Cas system comprising
one or more vectors for delivery to a eukaryotic cell, wherein the
vector(s) encode(s): (i) a CRISPR enzyme; (ii) a first guide RNA
capable of hybridizing to a target sequence in the cell; (iii) a
second guide RNA capable of hybridizing to one or more target
sequence(s) in the vector which encodes the CRISPR enzyme; (iv) at
least one tracr mate sequence; and (v) at least one tracr sequence,
The first and second complexes can use the same tracr and tracr
mate, thus differing only by the guide sequence, wherein, when
expressed within the cell: the first guide RNA directs
sequence-specific binding of a first CRISPR complex to the target
sequence in the cell; the second guide RNA directs
sequence-specific binding of a second CRISPR complex to the target
sequence in the vector which encodes the CRISPR enzyme; the CRISPR
complexes comprise (a) a tracr mate sequence hybridised to a tracr
sequence and (b) a CRISPR enzyme bound to a guide RNA, such that a
guide RNA can hybridize to its target sequence; and the second
CRISPR complex inactivates the CRISPR-Cas system to prevent
continued expression of the CRISPR enzyme by the cell.
[0493] Further characteristics of the vector(s), the encoded
enzyme, the guide sequences, etc. are disclosed elsewhere herein.
For instance, one or both of the guide sequence(s) can be part of a
chiRNA sequence which provides the guide, tracr mate and tracr
sequences within a single RNA, such that the system can encode (i)
a CRISPR enzyme; (ii) a first chiRNA comprising a sequence capable
of hybridizing to a first target sequence in the cell, a first
tracr mate sequence, and a first tracr sequence; (iii) a second
guide RNA capable of hybridizing to the vector which encodes the
CRISPR enzyme, a second tracr mate sequence, and a second tracr
sequence. Similarly, the enzyme can include one or more NLS,
etc.
[0494] The various coding sequences (CRISPR enzyme, guide RNAs,
tracr and tracr mate) can be included on a single vector or on
multiple vectors. For instance, it is possible to encode the enzyme
on one vector and the various RNA sequences on another vector, or
to encode the enzyme and one chiRNA on one vector, and the
remaining chiRNA on another vector, or any other permutation. In
general, a system using a total of one or two different vectors is
preferred.
[0495] Where multiple vectors are used, it is possible to deliver
them in unequal numbers, and ideally with an excess of a vector
which encodes the first guide RNA relative to the second guide RNA,
thereby assisting in delaying final inactivation of the CRISPR
system until genome editing has had a chance to occur.
[0496] The first guide RNA can target any target sequence of
interest within a genome, as described elsewhere herein. The second
guide RNA targets a sequence within the vector which encodes the
CRISPR Cas9 enzyme, and thereby inactivates the enzyme's expression
from that vector. Thus the target sequence in the vector must be
capable of inactivating expression. Suitable target sequences can
be, for instance, near to or within the translational start codon
for the Cas9 coding sequence, in a non-coding sequence in the
promoter driving expression of the non-coding RNA elements, within
the promoter driving expression of the Cas9 gene, within 100 bp of
the ATG translational start codon in the Cas9 coding sequence,
and/or within the inverted terminal repeat (iTR) of a viral
delivery vector, e.g., in the AAV genome. A double stranded break
near this region can induce a frame shift in the Cas9 coding
sequence, causing a loss of protein expression. An alternative
target sequence for the "self-inactivating" guide RNA would aim to
edit/inactivate regulatory regions/sequences needed for the
expression of the CRISPR-Cas9 system or for the stability of the
vector. For instance, if the promoter for the Cas9 coding sequence
is disrupted then transcription can be inhibited or prevented.
Similarly, if a vector includes sequences for replication,
maintenance or stability then it is possible to target these. For
instance, in a AAV vector a useful target sequence is within the
iTR. Other useful sequences to target can be promoter sequences,
polyadenylation sites, etc.
[0497] Furthermore, if the guide RNAs are expressed in array
format, the "self-inactivating" guide RNAs that target both
promoters simultaneously will result in the excision of the
intervening nucleotides from within the CRISPR-Cas expression
construct, effectively leading to its complete inactivation.
Similarly, excision of the intervening nucleotides will result
where the guide RNAs target both ITRs, or targets two or more other
CRISPR-Cas components simultaneously. Self-inactivation as
explained herein is applicable, in general, with CRISPR-Cas9
systems in order to provide regulation of the CRISPR-Cas9. For
example, self-inactivation as explained herein may be applied to
the CRISPR repair of mutations, for example expansion disorders, as
explained herein. As a result of this self-inactivation, CRISPR
repair is only transiently active.
[0498] Addition of non-targeting nucleotides to the 5' end (e.g.
1-10 nucleotides, preferably 1-5 nucleotides) of the
"self-inactivating" guide RNA can be used to delay its processing
and/or modify its efficiency as a means of ensuring editing at the
targeted genomic locus prior to CRISPR-Cas9 shutdown.
[0499] In one aspect of the self-inactivating AAV-CRISPR-Cas9
system, plasmids that co-express one or more sgRNA targeting
genomic sequences of interest (e.g. 1-2, 1-5, 1-10, 1-15, 1-20,
1-30) may be established with "self-inactivating" sgRNAs that
target an SpCas9 sequence at or near the engineered ATG start site
(e.g. within 5 nucleotides, within 15 nucleotides, within 30
nucleotides, within 50 nucleotides, within 100 nucleotides). A
regulatory sequence in the U6 promoter region can also be targeted
with an sgRNA. The U6-driven sgRNAs may be designed in an array
format such that multiple sgRNA sequences can be simultaneously
released. When first delivered into target tissue/cells (left cell)
sgRNAs begin to accumulate while Cas9 levels rise in the nucleus.
Cas9 complexes with all of the sgRNAs to mediate genome editing and
self-inactivation of the CRISPR-Cas9 plasmids.
[0500] One aspect of a self-inactivating CRISPR-Cas9 system is
expression of singly or in tandam array format from 1 up to 4 or
more different guide sequences; e.g. up to about 20 or about 30
guides sequences. Each individual self inactivating guide sequence
may target a different target. Such may be processed from, e.g. one
chimeric pol3 transcript. Pol3 promoters such as U6 or H1 promoters
may be used. Pol2 promoters such as those mentioned throughout
herein. Inverted terminal repeat (iTR) sequences may flank the Pol3
promoter-sgRNA(s)-Pol2 promoter-Cas9.
[0501] One aspect of a chimeric, tandem array transcript is that
one or more guide(s) edit the one or more target(s) while one or
more self inactivating guides inactivate the CRISPR/Cas9 system.
Thus, for example, the described CRISPR-Cas9 system for repairing
expansion disorders may be directly combined with the
self-inactivating CRISPR-Cas9 system described herein. Such a
system may, for example, have two guides directed to the target
region for repair as well as at least a third guide directed to
self-inactivation of the CRISPR-Cas9. Reference is made to
Application Ser. No. PCT/US2014/069897, entitled "Compositions And
Methods Of Use Of Crispr-Cas Systems In Nucleotide Repeat
Disorders," published Dec. 12, 2014 as WO/2015/089351.
[0502] The guideRNA may be a control guide. For example it may be
engineered to target a nucleic acid sequence encoding the CRISPR
Enzyme itself, as described in US2015232881A1, the disclosure of
which is hereby incorporated by reference. In some embodiments, a
system or composition may be provided with just the guideRNA
engineered to target the nucleic acid sequence encoding the CRISPR
Enzyme. In addition, the system or composition may be provided with
the guideRNA engineered to target the nucleic acid sequence
encoding the CRISPR Enzyme, as well as nucleic acid sequence
encoding the CRISPR Enzyme and, optionally a second guide RNA and,
further optionally, a repair template. The second guideRNA may be
the primary target of the CRISPR system or composition (such a
therapeutic, diagnostic, knock out etc. as defined herein). In this
way, the system or composition is self-inactivating. This is
exemplified in relation to Cas9 in US2015232881A1 (also published
as WO2015070083 (A1), referenced elsewhere herein).
Kits
[0503] In one aspect, the invention provides kits containing any
one or more of the elements disclosed in the above methods and
compositions. In some embodiments, the kit comprises a vector
system and instructions for using the kit. In some embodiments, the
vector system comprises (a) a first regulatory element operably
linked to a tracr mate sequence and one or more insertion sites for
inserting a guide sequence upstream of the tracr mate sequence,
wherein when expressed, the guide sequence directs
sequence-specific binding of a CRISPR complex to a target sequence
in a eukaryotic cell, wherein the CRISPR complex comprises a CRISPR
enzyme complexed with (1) the guide sequence that is hybridized to
the target sequence, and (2) the tracr mate sequence that is
hybridized to the tracr sequence; and/or (b) a second regulatory
element operably linked to an enzyme-coding sequence encoding said
CRISPR enzyme comprising a nuclear localization sequence. Elements
may be provide individually or in combinations, and may be provided
in any suitable container, such as a vial, a bottle, or a tube. The
kits may include the sgRNA and the unbound protector strand as
described herein. The kits may include the sgRNA with the protector
strand bound to at least partially to the guide sequence (i.e.
pgRNA). Thus the kits may include the pgRNA in the form of a
partially double stranded nucleotide sequence as described here. In
some embodiments, the kit includes instructions in one or more
languages, for example in more than one language. The instructions
may be specific to the applications and methods described
herein.
[0504] In some embodiments, a kit comprises one or more reagents
for use in a process utilizing one or more of the elements
described herein. Reagents may be provided in any suitable
container. For example, a kit may provide one or more reaction or
storage buffers. Reagents may be provided in a form that is usable
in a particular assay, or in a form that requires addition of one
or more other components before use (e.g. in concentrate or
lyophilized form). A buffer can be any buffer, including but not
limited to a sodium carbonate buffer, a sodium bicarbonate buffer,
a borate buffer, a Tris buffer, a MOPS buffer, a HEPES buffer, and
combinations thereof. In some embodiments, the buffer is alkaline.
In some embodiments, the buffer has a pH from about 7 to about 10.
In some embodiments, the kit comprises one or more oligonucleotides
corresponding to a guide sequence for insertion into a vector so as
to operably link the guide sequence and a regulatory element. In
some embodiments, the kit comprises a homologous recombination
template polynucleotide. In some embodiments, the kit comprises one
or more of the vectors and/or one or more of the polynucleotides
described herein. The kit may advantageously allow to provide all
elements of the systems of the invention.
[0505] In one aspect, the invention provides methods for using one
or more elements of a CRISPR system. The CRISPR complex of the
invention provides an effective means for modifying a target
polynucleotide. The CRISPR complex of the invention has a wide
variety of utility including modifying (e.g., deleting, inserting,
translocating, inactivating, activating) a target polynucleotide in
a multiplicity of cell types. As such the CRISPR complex of the
invention has a broad spectrum of applications in, e.g., gene
therapy, drug screening, disease diagnosis, and prognosis. An
exemplary CRISPR complex comprises a CRISPR effector protein
complexed with a guide sequence hybridized to a target sequence
within the target polynucleotide. In certain embodiments, a direct
repeat sequence is linked to the guide sequence.
[0506] In one embodiment, this invention provides a method of
cleaving a target polynucleotide. The method comprises modifying a
target polynucleotide using a CRISPR complex that binds to the
target polynucleotide and effect cleavage of said target
polynucleotide. Typically, the CRISPR complex of the invention,
when introduced into a cell, creates a break (e.g., a single or a
double strand break) in the genome sequence. For example, the
method can be used to cleave a disease gene in a cell.
[0507] The break created by the CRISPR complex can be repaired by a
repair processes such as the error prone non-homologous end joining
(NHEJ) pathway or the high fidelity homology directed repair (HDR).
During these repair process, an exogenous polynucleotide template
can be introduced into the genome sequence. In some methods, the
HDR process is used to modify genome sequence. For example, an
exogenous polynucleotide template comprising a sequence to be
integrated flanked by an upstream sequence and a downstream
sequence is introduced into a cell. The upstream and downstream
sequences share sequence similarity with either side of the site of
integration in the chromosome.
[0508] Where desired, a donor polynucleotide can be DNA, e.g., a
DNA plasmid, a bacterial artificial chromosome (BAC), a yeast
artificial chromosome (YAC), a viral vector, a linear piece of DNA,
a PCR fragment, a naked nucleic acid, or a nucleic acid complexed
with a delivery vehicle such as a liposome or poloxamer.
[0509] The exogenous polynucleotide template comprises a sequence
to be integrated (e.g., a mutated gene). The sequence for
integration may be a sequence endogenous or exogenous to the cell.
Examples of a sequence to be integrated include polynucleotides
encoding a protein or a non-coding RNA (e.g., a microRNA). Thus,
the sequence for integration may be operably linked to an
appropriate control sequence or sequences. Alternatively, the
sequence to be integrated may provide a regulatory function.
[0510] The upstream and downstream sequences in the exogenous
polynucleotide template are selected to promote recombination
between the chromosomal sequence of interest and the donor
polynucleotide. The upstream sequence is a nucleic acid sequence
that shares sequence similarity with the genome sequence upstream
of the targeted site for integration. Similarly, the downstream
sequence is a nucleic acid sequence that shares sequence similarity
with the chromosomal sequence downstream of the targeted site of
integration. The upstream and downstream sequences in the exogenous
polynucleotide template can have 75%, 80%, 85%, 90%, 95%, or 100%
sequence identity with the targeted genome sequence. Preferably,
the upstream and downstream sequences in the exogenous
polynucleotide template have about 95%, 96%, 97%, 98%, 99%, or 100%
sequence identity with the targeted genome sequence. In some
methods, the upstream and downstream sequences in the exogenous
polynucleotide template have about 99% or 100% sequence identity
with the targeted genome sequence.
[0511] An upstream or downstream sequence may comprise from about
20 bp to about 2500 bp, for example, about 50, 100, 200, 300, 400,
500, 600, 700, 800, 900, 1000, 1100, 1200, 1300, 1400, 1500, 1600,
1700, 1800, 1900, 2000, 2100, 2200, 2300, 2400, or 2500 bp. In some
methods, the exemplary upstream or downstream sequence have about
200 bp to about 2000 bp, about 600 bp to about 1000 bp, or more
particularly about 700 bp to about 1000 bp.
[0512] In some methods, the exogenous polynucleotide template may
further comprise a marker. Such a marker may make it easy to screen
for targeted integrations. Examples of suitable markers include
restriction sites, fluorescent proteins, or selectable markers. The
exogenous polynucleotide template of the invention can be
constructed using recombinant techniques (see, for example,
Sambrook et al., 2001 and Ausubel et al., 1996).
[0513] In an exemplary method for modifying a target polynucleotide
by integrating an exogenous polynucleotide template, a double
stranded break is introduced into the genome sequence by the CRISPR
complex, the break is repaired via homologous recombination an
exogenous polynucleotide template such that the template is
integrated into the genome. The presence of a double-stranded break
facilitates integration of the template.
[0514] In other embodiments, this invention provides a method of
modifying expression of a polynucleotide in a eukaryotic cell. The
method comprises increasing or decreasing expression of a target
polynucleotide by using a CRISPR complex that binds to the
polynucleotide.
[0515] In some methods, a target polynucleotide can be inactivated
to effect the modification of the expression in a cell. For
example, upon the binding of a CRISPR complex to a target sequence
in a cell, the target polynucleotide is inactivated such that the
sequence is not transcribed, the coded protein is not produced, or
the sequence does not function as the wild-type sequence does. For
example, a protein or microRNA coding sequence may be inactivated
such that the protein is not produced.
[0516] In some methods, a control sequence can be inactivated such
that it no longer functions as a control sequence. As used herein,
"control sequence" refers to any nucleic acid sequence that effects
the transcription, translation, or accessibility of a nucleic acid
sequence. Examples of a control sequence include, a promoter, a
transcription terminator, and an enhancer are control sequences.
The inactivated target sequence may include a deletion mutation
(i.e., deletion of one or more nucleotides), an insertion mutation
(i.e., insertion of one or more nucleotides), or a nonsense
mutation (i.e., substitution of a single nucleotide for another
nucleotide such that a stop codon is introduced). In some methods,
the inactivation of a target sequence results in "knockout" of the
target sequence.
Modifying a Target with CRISPR-Cas9 System or Complex
[0517] In one aspect, the invention provides for methods of
modifying a target polynucleotide in a eukaryotic cell, which may
be in vivo, ex vivo or in vitro. In some embodiments, the method
comprises sampling a cell or population of cells from a human or
non-human animal or plant (including micro-algae), and modifying
the cell or cells. Culturing may occur at any stage ex vivo. The
cell or cells may even be re-introduced into the non-human animal
or plant. For re-introduced cells it is particularly preferred that
the cells are stem cells.
[0518] In some embodiments, the method comprises allowing a CRISPR
complex to bind to the target polynucleotide to effect cleavage of
said target polynucleotide thereby modifying the target
polynucleotide, wherein the CRISPR complex comprises a CRISPR
enzyme complexed with a guide sequence hybridized or hybridizable
to a target sequence within said target polynucleotide, wherein
said guide sequence is linked to a tracr mate sequence which in
turn hybridizes to a tracr sequence.
[0519] In one aspect, the invention provides a method of modifying
expression of a polynucleotide in a eukaryotic cell. In some
embodiments, the method comprises allowing a CRISPR complex to bind
to the polynucleotide such that said binding results in increased
or decreased expression of said polynucleotide; wherein the CRISPR
complex comprises a CRISPR enzyme complexed with a guide sequence
hybridized or hybridizable to a target sequence within said
polynucleotide, wherein said guide sequence is linked to a tracr
mate sequence which in turn hybridizes to a tracr sequence. Similar
considerations and conditions apply as above for methods of
modifying a target polynucleotide. In fact, these sampling,
culturing and re-introduction options apply across the aspects of
the present invention.
[0520] Indeed, in any aspect of the invention, the CRISPR complex
may comprise a CRISPR enzyme complexed with a guide sequence
hybridized or hybridizable to a target sequence, wherein said guide
sequence may be linked to a tracr mate sequence which in turn may
hybridize to a tracr sequence. Similar considerations and
conditions apply as above for methods of modifying a target
polynucleotide.
[0521] In plants, pathogens are often host-specific. For example,
Fusarium oxysporum f. sp. lycopersici causes tomato wilt but
attacks only tomato, and F. oxysporum f. dianthii Puccinia graminis
f sp. tritici attacks only wheat. Plants have existing and induced
defenses to resist most pathogens. Mutations and recombination
events across plant generations lead to genetic variability that
gives rise to susceptibility, especially as pathogens reproduce
with more frequency than plants. In plants there can be non-host
resistance, e.g., the host and pathogen are incompatible. There can
also be Horizontal Resistance, e.g., partial resistance against all
races of a pathogen, typically controlled by many genes and
Vertical Resistance, e.g., complete resistance to some races of a
pathogen but not to other races, typically controlled by a few
genes. In a Gene-for-Gene level, plants and pathogens evolve
together, and the genetic changes in one balance changes in other.
Accordingly, using Natural Variability, breeders combine most
useful genes for Yield, Quality, Uniformity, Hardiness, Resistance.
The sources of resistance genes include native or foreign
Varieties, Heirloom Varieties, Wild Plant Relatives, and Induced
Mutations, e.g., treating plant material with mutagenic agents.
Using the present invention, plant breeders are provided with a new
tool to induce mutations. Accordingly, one skilled in the art can
analyze the genome of sources of resistance genes, and in Varieties
having desired characteristics or traits employ the present
invention to induce the rise of resistance genes, with more
precision than previous mutagenic agents and hence accelerate and
improve plant breeding programs.
[0522] Similar considerations and conditions apply as above for
methods of modifying a target polynucleotide. Thus in any of the
non-naturally-occurring CRISPR enzymes described herein comprise at
least one modification and whereby the enzyme has certain improved
capabilities. In particular, any of the enzymes are capable of
forming a CRISPR complex with a guide RNA. When such a complex
forms, the guide RNA is capable of binding to a target
polynucleotide sequence and the enzyme is capable of modifying a
target locus. In addition, the enzyme in the CRISPR complex has
reduced capability of modifying one or more off-target loci as
compared to an unmodified enzyme.
[0523] In addition, the modified CRISPR enzymes described herein
encompass enzymes whereby in the CRISPR complex the enzyme has
increased capability of modifying the one or more target loci as
compared to an unmodified enzyme. Such function may be provided
separate to or provided in combination with the above-described
function of reduced capability of modifying one or more off-target
loci. Any such enzymes may be provided with any of the further
modifications to the CRISPR enzyme as described herein, such as in
combination with any activity provided by one or more associated
heterologous functional domains, any further mutations to reduce
nuclease activity and the like.
[0524] In advantageous embodiments of the invention, the modified
CRISPR enzyme is provided with reduced capability of modifying one
or more off-target loci as compared to an unmodified enzyme and
increased capability of modifying the one or more target loci as
compared to an unmodified enzyme. In combination with further
modifications to the enzyme, significantly enhanced specificity may
be achieved. For example, combination of such advantageous
embodiments with one or more additional mutations is provided
wherein the one or more additional mutations are in one or more
catalytically active domains. Such further catalytic mutations may
confer nickase functionality as described in detail elsewhere
herein. In such enzymes, enhanced specificity may be achieved due
to an improved specificity in terms of enzyme activity.
[0525] Modifications to reduce off-target effects and/or enhance
on-target effects as described above may be made to amino acid
residues located in a positively-charged region/groove situated
between the RuvC-III and HNH domains. It will be appreciated that
any of the functional effects described above may be achieved by
modification of amino acids within the aforementioned groove but
also by modification of amino acids adjacent to or outside of that
groove.
[0526] Additional functionalities which may be engineered into
modified CRISPR enzymes as described herein include the following.
1. modified CRISPR enzymes that disrupt DNA:protein interactions
without affecting protein tertiary or secondary structure. This
includes residues that contact any part of the RNA:DNA duplex. 2.
modified CRISPR enzymes that weaken intra-protein interactions
holding Cas9 in conformation essential for nuclease cutting in
response to DNA binding (on or off target). For example: a
modification that mildly inhibits, but still allows, the nuclease
conformation of the HNH domain (positioned at the scissile
phosphate). 3. modified CRISPR enzymes that strengthen
intra-protein interactions holding Cas9 in a conformation
inhibiting nuclease activity in response to DNA binding (on or off
targets). For example: a modification that stabilizes the HNH
domain in a conformation away from the scissile phosphate. Any such
additional functional enhancement may be provided in combination
with any other modification to the CRISPR enzyme as described in
detail elsewhere herein.
[0527] Any of the herein described improved functionalities may be
made to any CRISPR enzyme, such as a Cas9 enzyme. Cas9 enzymes
described herein are derived from Cas9 enzymes from S. pyogenes and
S. aureus. However, it will be appreciated that any of the
functionalities described herein may be engineered into Cas9
enzymes from other orthologs, including chimeric enzymes comprising
fragments from multiple orthologs.
Nucleic Acids, Amino Acids and Proteins, Regulatory Sequences,
Vectors, Etc
[0528] The invention uses nucleic acids to bind target DNA
sequences. This is advantageous as nucleic acids are much easier
and cheaper to produce than proteins, and the specificity can be
varied according to the length of the stretch where homology is
sought. Complex 3-D positioning of multiple fingers, for example is
not required. The terms "polynucleotide", "nucleotide", "nucleotide
sequence", "nucleic acid" and "oligonucleotide" are used
interchangeably. They refer to a polymeric form of nucleotides of
any length, either deoxyribonucleotides or ribonucleotides, or
analogs thereof. Polynucleotides may have any three dimensional
structure, and may perform any function, known or unknown. The
following are non-limiting examples of polynucleotides: coding or
non-coding regions of a gene or gene fragment, loci (locus) defined
from linkage analysis, exons, introns, messenger RNA (mRNA),
transfer RNA, ribosomal RNA, short interfering RNA (siRNA),
short-hairpin RNA (shRNA), micro-RNA (miRNA), ribozymes, cDNA,
recombinant polynucleotides, branched polynucleotides, plasmids,
vectors, isolated DNA of any sequence, isolated RNA of any
sequence, nucleic acid probes, and primers. The term also
encompasses nucleic-acid-like structures with synthetic backbones,
see, e.g., Eckstein, 1991; Baserga et al., 1992; Milligan, 1993; WO
97/03211; WO 96/39154; Mata, 1997; Strauss-Soukup, 1997; and
Samstag, 1996. A polynucleotide may comprise one or more modified
nucleotides, such as methylated nucleotides and nucleotide analogs.
If present, modifications to the nucleotide structure may be
imparted before or after assembly of the polymer. The sequence of
nucleotides may be interrupted by non-nucleotide components. A
polynucleotide may be further modified after polymerization, such
as by conjugation with a labeling component.
[0529] In aspects of the invention the terms "chimeric RNA",
"chimeric guide RNA", "guide RNA", "single guide RNA" and
"synthetic guide RNA" are used interchangeably and refer to the
polynucleotide sequence comprising the guide sequence, the tracr
sequence and the tracr mate sequence. The term "guide sequence"
refers to the about 20 bp sequence within the guide RNA that
specifies the target site and may be used interchangeably with the
terms "guide" or "spacer". The term "tracr mate sequence" may also
be used interchangeably with the term "direct repeat(s)".
[0530] As used herein the term "wild type" is a term of the art
understood by skilled persons and means the typical form of an
organism, strain, gene or characteristic as it occurs in nature as
distinguished from mutant or variant forms. A "wild type" can be a
base line.
[0531] As used herein the term "variant" should be taken to mean
the exhibition of qualities that have a pattern that deviates from
what occurs in nature.
[0532] The terms "non-naturally occurring" or "engineered" are used
interchangeably and indicate the involvement of the hand of man.
The terms, when referring to nucleic acid molecules or polypeptides
mean that the nucleic acid molecule or the polypeptide is at least
substantially free from at least one other component with which
they are naturally associated in nature and as found in nature.
[0533] "Complementarity" refers to the ability of a nucleic acid to
form hydrogen bond(s) with another nucleic acid sequence by either
traditional Watson-Crick base pairing or other non-traditional
types. A percent complementarity indicates the percentage of
residues in a nucleic acid molecule which can form hydrogen bonds
(e.g., Watson-Crick base pairing) with a second nucleic acid
sequence (e.g., 5, 6, 7, 8, 9, 10 out of 10 being 50%, 60%, 70%,
80%, 90%, and 100% complementary). "Perfectly complementary" means
that all the contiguous residues of a nucleic acid sequence will
hydrogen bond with the same number of contiguous residues in a
second nucleic acid sequence. "Substantially complementary" as used
herein refers to a degree of complementarity that is at least 60%,
65%, 70%, 75%, 80%, 85%, 90%, 95%, 97%, 98%, 99%, or 100% over a
region of 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22,
23, 24, 25, 30, 35, 40, 45, 50, or more nucleotides, or refers to
two nucleic acids that hybridize under stringent conditions.
[0534] As used herein, "stringent conditions" for hybridization
refer to conditions under which a nucleic acid having
complementarity to a target sequence predominantly hybridizes with
the target sequence, and substantially does not hybridize to
non-target sequences. Stringent conditions are generally
sequence-dependent, and vary depending on a number of factors. In
general, the longer the sequence, the higher the temperature at
which the sequence specifically hybridizes to its target sequence.
Non-limiting examples of stringent conditions are described in
detail in Tijssen (1993), Laboratory Techniques In Biochemistry And
Molecular Biology-Hybridization With Nucleic Acid Probes Part I,
Second Chapter "Overview of principles of hybridization and the
strategy of nucleic acid probe assay", Elsevier, N.Y. Where
reference is made to a polynucleotide sequence, then complementary
or partially complementary sequences are also envisaged. These are
preferably capable of hybridizing to the reference sequence under
highly stringent conditions. Generally, in order to maximize the
hybridization rate, relatively low-stringency hybridization
conditions are selected: about 20 to 25.degree. C. lower than the
thermal melting point (T.sub.m). The T. is the temperature at which
50% of specific target sequence hybridizes to a perfectly
complementary probe in solution at a defined ionic strength and pH.
Generally, in order to require at least about 85% nucleotide
complementarity of hybridized sequences, highly stringent washing
conditions are selected to be about 5 to 15.degree. C. lower than
the T.sub.m. In order to require at least about 70% nucleotide
complementarity of hybridized sequences, moderately-stringent
washing conditions are selected to be about 15 to 30.degree. C.
lower than the T. Highly permissive (very low stringency) washing
conditions may be as low as 50.degree. C. below the T.sub.m,
allowing a high level of mis-matching between hybridized sequences.
Those skilled in the art will recognize that other physical and
chemical parameters in the hybridization and wash stages can also
be altered to affect the outcome of a detectable hybridization
signal from a specific level of homology between target and probe
sequences. Preferred highly stringent conditions comprise
incubation in 50% formamide, 5.times.SSC, and 1% SDS at 42.degree.
C., or incubation in 5.times.SSC and 1% SDS at 65.degree. C., with
wash in 0.2.times.SSC and 0.1% SDS at 65.degree. C.
[0535] "Hybridization" refers to a reaction in which one or more
polynucleotides react to form a complex that is stabilized via
hydrogen bonding between the bases of the nucleotide residues. The
hydrogen bonding may occur by Watson Crick base pairing, Hoogstein
binding, or in any other sequence specific manner. The complex may
comprise two strands forming a duplex structure, three or more
strands forming a multi stranded complex, a single self hybridizing
strand, or any combination of these. A hybridization reaction may
constitute a step in a more extensive process, such as the
initiation of PCR, or the cleavage of a polynucleotide by an
enzyme. A sequence capable of hybridizing with a given sequence is
referred to as the "complement" of the given sequence.
[0536] As used herein, the term "genomic locus" or "locus" (plural
loci) is the specific location of a gene or DNA sequence on a
chromosome. A "gene" refers to stretches of DNA or RNA that encode
a polypeptide or an RNA chain that has functional role to play in
an organism and hence is the molecular unit of heredity in living
organisms. For the purpose of this invention it may be considered
that genes include regions which regulate the production of the
gene product, whether or not such regulatory sequences are adjacent
to coding and/or transcribed sequences. Accordingly, a gene
includes, but is not necessarily limited to, promoter sequences,
terminators, translational regulatory sequences such as ribosome
binding sites and internal ribosome entry sites, enhancers,
silencers, insulators, boundary elements, replication origins,
matrix attachment sites and locus control regions. As used herein,
"expression of a genomic locus" or "gene expression" is the process
by which information from a gene is used in the synthesis of a
functional gene product. The products of gene expression are often
proteins, but in non-protein coding genes such as rRNA genes or
tRNA genes, the product is functional RNA. The process of gene
expression is used by all known life--eukaryotes (including
multicellular organisms), prokaryotes (bacteria and archaea) and
viruses to generate functional products to survive. As used herein
"expression" of a gene or nucleic acid encompasses not only
cellular gene expression, but also the transcription and
translation of nucleic acid(s) in cloning systems and in any other
context.
[0537] As used herein, "expression" refers to the process by which
a polynucleotide is transcribed from a DNA template (such as into
and mRNA or other RNA transcript) and/or the process by which a
transcribed mRNA is subsequently translated into peptides,
polypeptides, or proteins. Transcripts and encoded polypeptides may
be collectively referred to as "gene product." If the
polynucleotide is derived from genomic DNA, expression may include
splicing of the mRNA in a eukaryotic cell.
[0538] The terms "polypeptide", "peptide" and "protein" are used
interchangeably herein to refer to polymers of amino acids of any
length. The polymer may be linear or branched, it may comprise
modified amino acids, and it may be interrupted by non amino acids.
The terms also encompass an amino acid polymer that has been
modified; for example, disulfide bond formation, glycosylation,
lipidation, acetylation, phosphorylation, or any other
manipulation, such as conjugation with a labeling component. As
used herein the term "amino acid" includes natural and/or unnatural
or synthetic amino acids, including glycine and both the D or L
optical isomers, and amino acid analogs and peptidomimetics.
[0539] As used herein, the term "domain" or "protein domain" refers
to a part of a protein sequence that may exist and function
independently of the rest of the protein chain. As described in
aspects of the invention, sequence identity is related to sequence
homology. Homology comparisons may be conducted by eye, or more
usually, with the aid of readily available sequence comparison
programs. These commercially available computer programs may
calculate percent (%) homology between two or more sequences and
may also calculate the sequence identity shared by two or more
amino acid or nucleic acid sequences.
[0540] In aspects of the invention the term "guide RNA", refers to
the polynucleotide sequence comprising one or more of a putative or
identified tracr sequence and a putative or identified crRNA
sequence or guide sequence. In particular embodiments, the "guide
RNA" comprises a putative or identified crRNA sequence or guide
sequence. In further embodiments, the guide RNA does not comprise a
putative or identified tracr sequence.
[0541] As used herein the term "wild type" is a term of the art
understood by skilled persons and means the typical form of an
organism, strain, gene or characteristic as it occurs in nature as
distinguished from mutant or variant forms. A "wild type" can be a
base line.
[0542] As used herein the term "variant" should be taken to mean
the exhibition of qualities that have a pattern that deviates from
what occurs in nature.
[0543] The terms "non-naturally occurring" or "engineered" are used
interchangeably and indicate the involvement of the hand of man.
The terms, when referring to nucleic acid molecules or polypeptides
mean that the nucleic acid molecule or the polypeptide is at least
substantially free from at least one other component with which
they are naturally associated in nature and as found in nature. In
all aspects and embodiments, whether they include these terms or
not, it will be understood that, preferably, the may be optional
and thus preferably included or not preferably not included.
Furthermore, the terms "non-naturally occurring" and "engineered"
may be used interchangeably and so can therefore be used alone or
in combination and one or other may replace mention of both
together. In particular, "engineered" is preferred in place of
"non-naturally occurring" or "non-naturally occurring and/or
engineered."
[0544] Sequence homologies may be generated by any of a number of
computer programs known in the art, for example BLAST or FASTA,
etc. A suitable computer program for carrying out such an alignment
is the GCG Wisconsin Bestfit package (University of Wisconsin,
U.S.A; Devereux et al., 1984, Nucleic Acids Research 12:387).
Examples of other software than may perform sequence comparisons
include, but are not limited to, the BLAST package (see Ausubel et
al., 1999 ibid--Chapter 18), FASTA (Atschul et al., 1990, J. Mol.
Biol., 403-410) and the GENEWORKS suite of comparison tools. Both
BLAST and FASTA are available for offline and online searching (see
Ausubel et al., 1999 ibid, pages 7-58 to 7-60). However it is
preferred to use the GCG Bestfit program. Percentage (%) sequence
homology may be calculated over contiguous sequences, i.e., one
sequence is aligned with the other sequence and each amino acid or
nucleotide in one sequence is directly compared with the
corresponding amino acid or nucleotide in the other sequence, one
residue at a time. This is called an "ungapped" alignment.
Typically, such ungapped alignments are performed only over a
relatively short number of residues. Although this is a very simple
and consistent method, it fails to take into consideration that,
for example, in an otherwise identical pair of sequences, one
insertion or deletion may cause the following amino acid residues
to be put out of alignment, thus potentially resulting in a large
reduction in % homology when a global alignment is performed.
Consequently, most sequence comparison methods are designed to
produce optimal alignments that take into consideration possible
insertions and deletions without unduly penalizing the overall
homology or identity score. This is achieved by inserting "gaps" in
the sequence alignment to try to maximize local homology or
identity. However, these more complex methods assign "gap
penalties" to each gap that occurs in the alignment so that, for
the same number of identical amino acids, a sequence alignment with
as few gaps as possible--reflecting higher relatedness between the
two compared sequences--may achieve a higher score than one with
many gaps. "Affinity gap costs" are typically used that charge a
relatively high cost for the existence of a gap and a smaller
penalty for each subsequent residue in the gap. This is the most
commonly used gap scoring system. High gap penalties may, of
course, produce optimized alignments with fewer gaps. Most
alignment programs allow the gap penalties to be modified. However,
it is preferred to use the default values when using such software
for sequence comparisons. For example, when using the GCG Wisconsin
Bestfit package the default gap penalty for amino acid sequences is
-12 for a gap and -4 for each extension. Calculation of maximum %
homology therefore first requires the production of an optimal
alignment, taking into consideration gap penalties. A suitable
computer program for carrying out such an alignment is the GCG
Wisconsin Bestfit package (Devereux et al., 1984 Nuc. Acids
Research 12 p387). Examples of other software than may perform
sequence comparisons include, but are not limited to, the BLAST
package (see Ausubel et al., 1999 Short Protocols in Molecular
Biology, 4.sup.th Ed.--Chapter 18), FASTA (Altschul et al., 1990 J
Mol. Biol. 403-410) and the GENEWORKS suite of comparison tools.
Both BLAST and FASTA are available for offline and online searching
(see Ausubel et al., 1999, Short Protocols in Molecular Biology,
pages 7-58 to 7-60). However, for some applications, it is
preferred to use the GCG Bestfit program. A new tool, called BLAST
2 Sequences is also available for comparing protein and nucleotide
sequences (see FEMS Microbiol Lett. 1999 174(2): 247-50; FEMS
Microbiol Lett. 1999 177(1): 187-8 and the website of the National
Center for Biotechnology information at the website of the National
Institutes for Health). Although the final % homology may be
measured in terms of identity, the alignment process itself is
typically not based on an all-or-nothing pair comparison. Instead,
a scaled similarity score matrix is generally used that assigns
scores to each pair-wise comparison based on chemical similarity or
evolutionary distance. An example of such a matrix commonly used is
the BLOSUM62 matrix--the default matrix for the BLAST suite of
programs. GCG Wisconsin programs generally use either the public
default values or a custom symbol comparison table, if supplied
(see user manual for further details). For some applications, it is
preferred to use the public default values for the GCG package, or
in the case of other software, the default matrix, such as
BLOSUM62. Alternatively, percentage homologies may be calculated
using the multiple alignment feature in DNASIS.TM. (Hitachi
Software), based on an algorithm, analogous to CLUSTAL (Higgins D G
& Sharp P M (1988), Gene 73(1), 237-244). Once the software has
produced an optimal alignment, it is possible to calculate %
homology, preferably % sequence identity. The software typically
does this as part of the sequence comparison and generates a
numerical result. The sequences may also have deletions, insertions
or substitutions of amino acid residues which produce a silent
change and result in a functionally equivalent substance.
Deliberate amino acid substitutions may be made on the basis of
similarity in amino acid properties (such as polarity, charge,
solubility, hydrophobicity, hydrophilicity, and/or the amphipathic
nature of the residues) and it is therefore useful to group amino
acids together in functional groups. Amino acids may be grouped
together based on the properties of their side chains alone.
However, it is more useful to include mutation data as well. The
sets of amino acids thus derived are likely to be conserved for
structural reasons. These sets may be described in the form of a
Venn diagram (Livingstone C. D. and Barton G. J. (1993) "Protein
sequence alignments: a strategy for the hierarchical analysis of
residue conservation" Comput. Appl. Biosci. 9: 745-756) (Taylor W.
R. (1986) "The classification of amino acid conservation" J. Theor.
Biol. 119; 205-218). Conservative substitutions may be made, for
example according to the table below which describes a generally
accepted Venn diagram grouping of amino acids.
TABLE-US-00012 Set Sub-set Hydrophobic FWYHKMILVAGC Aromatic FWYH
Aliphatic ILV Polar WYHKREDCSTNQ Charged HKRED Positively HKR
charged Negatively ED charged Small VCAGSPTND Tiny AGS
[0545] The terms "subject," "individual," and "patient" are used
interchangeably herein to refer to a vertebrate, preferably a
mammal, more preferably a human. Mammals include, but are not
limited to, murines, simians, humans, farm animals, sport animals,
and pets. Tissues, cells and their progeny of a biological entity
obtained in vivo or cultured in vitro are also encompassed.
[0546] The terms "therapeutic agent", "therapeutic capable agent"
or "treatment agent" are used interchangeably and refer to a
molecule or compound that confers some beneficial effect upon
administration to a subject. The beneficial effect includes
enablement of diagnostic determinations; amelioration of a disease,
symptom, disorder, or pathological condition; reducing or
preventing the onset of a disease, symptom, disorder or condition;
and generally counteracting a disease, symptom, disorder or
pathological condition.
[0547] As used herein, "treatment" or "treating," or "palliating"
or "ameliorating" are used interchangeably. These terms refer to an
approach for obtaining beneficial or desired results including but
not limited to a therapeutic benefit and/or a prophylactic benefit.
By therapeutic benefit is meant any therapeutically relevant
improvement in or effect on one or more diseases, conditions, or
symptoms under treatment. For prophylactic benefit, the
compositions may be administered to a subject at risk of developing
a particular disease, condition, or symptom, or to a subject
reporting one or more of the physiological symptoms of a disease,
even though the disease, condition, or symptom may not have yet
been manifested.
[0548] The term "effective amount" or "therapeutically effective
amount" refers to the amount of an agent that is sufficient to
effect beneficial or desired results. The therapeutically effective
amount may vary depending upon one or more of: the subject and
disease condition being treated, the weight and age of the subject,
the severity of the disease condition, the manner of administration
and the like, which can readily be determined by one of ordinary
skill in the art. The term also applies to a dose that will provide
an image for detection by any one of the imaging methods described
herein. The specific dose may vary depending on one or more of: the
particular agent chosen, the dosing regimen to be followed, whether
it is administered in combination with other compounds, timing of
administration, the tissue to be imaged, and the physical delivery
system in which it is carried.
[0549] The practice of the present invention employs, unless
otherwise indicated, conventional techniques of immunology,
biochemistry, chemistry, molecular biology, microbiology, cell
biology, genomics and recombinant DNA, which are within the skill
of the art. See Sambrook, Fritsch and Maniatis, MOLECULAR CLONING:
A LABORATORY MANUAL, 2nd edition (1989); CURRENT PROTOCOLS IN
MOLECULAR BIOLOGY (F. M. Ausubel, et al. eds., (1987)); the series
METHODS IN ENZYMOLOGY (Academic Press, Inc.): PCR 2: A PRACTICAL
APPROACH (M. J. MacPherson, B. D. Hames and G. R. Taylor eds.
(1995)), Harlow and Lane, eds. (1988) ANTIBODIES, A LABORATORY
MANUAL, and ANIMAL CELL CULTURE (R. I. Freshney, ed. (1987)).
[0550] Several aspects of the invention relate to vector systems
comprising one or more vectors, or vectors as such. Vectors can be
designed for expression of CRISPR transcripts (e.g. nucleic acid
transcripts, proteins, or enzymes) in prokaryotic or eukaryotic
cells. For example, CRISPR transcripts can be expressed in
bacterial cells such as Escherichia coli, insect cells (using
baculovirus expression vectors), yeast cells, or mammalian cells.
Suitable host cells are discussed further in Goeddel, GENE
EXPRESSION TECHNOLOGY: METHODS IN ENZYMOLOGY 185, Academic Press,
San Diego, Calif. (1990). Alternatively, the recombinant expression
vector can be transcribed and translated in vitro, for example
using T7 promoter regulatory sequences and T7 polymerase.
[0551] Embodiments of the invention include sequences (both
polynucleotide or polypeptide) which may comprise homologous
substitution (substitution and replacement are both used herein to
mean the interchange of an existing amino acid residue or
nucleotide, with an alternative residue or nucleotide) that may
occur i.e., like-for-like substitution in the case of amino acids
such as basic for basic, acidic for acidic, polar for polar, etc.
Non-homologous substitution may also occur i.e., from one class of
residue to another or alternatively involving the inclusion of
unnatural amino acids such as ornithine (hereinafter referred to as
Z), diaminobutyric acid ornithine (hereinafter referred to as B),
norleucine ornithine (hereinafter referred to as O), pyriylalanine,
thienylalanine, naphthylalanine and phenylglycine. Variant amino
acid sequences may include suitable spacer groups that may be
inserted between any two amino acid residues of the sequence
including alkyl groups such as methyl, ethyl or propyl groups in
addition to amino acid spacers such as glycine or .beta.-alanine
residues. A further form of variation, which involves the presence
of one or more amino acid residues in peptoid form, may be well
understood by those skilled in the art. For the avoidance of doubt,
"the peptoid form" is used to refer to variant amino acid residues
wherein the .alpha.-carbon substituent group is on the residue's
nitrogen atom rather than the .alpha.-carbon. Processes for
preparing peptides in the peptoid form are known in the art, for
example Simon R J et al., PNAS (1992) 89(20), 9367-9371 and Horwell
D C, Trends Biotechnol. (1995) 13(4), 132-134.
[0552] Homology modelling: Corresponding residues in other Cas9
orthologs can be identified by the methods of Zhang et al., 2012
(Nature; 490(7421): 556-60) and Chen et al., 2015 (PLoS Comput
Biol; 11(5): e1004248)--a computational protein-protein interaction
(PPI) method to predict interactions mediated by domain-motif
interfaces. PrePPI (Predicting PPI), a structure based PPI
prediction method, combines structural evidence with non-structural
evidence using a Bayesian statistical framework. The method
involves taking a pair a query proteins and using structural
alignment to identify structural representatives that correspond to
either their experimentally determined structures or homology
models. Structural alignment is further used to identify both close
and remote structural neighbors by considering global and local
geometric relationships. Whenever two neighbors of the structural
representatives form a complex reported in the Protein Data Bank,
this defines a template for modelling the interaction between the
two query proteins. Models of the complex are created by
superimposing the representative structures on their corresponding
structural neighbor in the template. This approach is further
described in Dey et al., 2013 (Prot Sci; 22: 359-66).
[0553] For purpose of this invention, amplification means any
method employing a primer and a polymerase capable of replicating a
target sequence with reasonable fidelity. Amplification may be
carried out by natural or recombinant DNA polymerases such as
TaqGold.TM., T7 DNA polymerase, Klenow fragment of E. coli DNA
polymerase, and reverse transcriptase. A preferred amplification
method is PCR.
[0554] In certain aspects the invention involves vectors. A used
herein, a "vector" is a tool that allows or facilitates the
transfer of an entity from one environment to another. It is a
replicon, such as a plasmid, phage, or cosmid, into which another
DNA segment may be inserted so as to bring about the replication of
the inserted segment. Generally, a vector is capable of replication
when associated with the proper control elements. In general, the
term "vector" refers to a nucleic acid molecule capable of
transporting another nucleic acid to which it has been linked.
Vectors include, but are not limited to, nucleic acid molecules
that are single-stranded, double-stranded, or partially
double-stranded; nucleic acid molecules that comprise one or more
free ends, no free ends (e.g., circular); nucleic acid molecules
that comprise DNA, RNA, or both; and other varieties of
polynucleotides known in the art. One type of vector is a
"plasmid," which refers to a circular double stranded DNA loop into
which additional DNA segments can be inserted, such as by standard
molecular cloning techniques. Another type of vector is a viral
vector, wherein virally-derived DNA or RNA sequences are present in
the vector for packaging into a virus (e.g., retroviruses,
replication defective retroviruses, adenoviruses, replication
defective adenoviruses, and adeno-associated viruses (AAVs)). Viral
vectors also include polynucleotides carried by a virus for
transfection into a host cell. Certain vectors are capable of
autonomous replication in a host cell into which they are
introduced (e.g., bacterial vectors having a bacterial origin of
replication and episomal mammalian vectors). Other vectors (e.g.,
non-episomal mammalian vectors) are integrated into the genome of a
host cell upon introduction into the host cell, and thereby are
replicated along with the host genome. Moreover, certain vectors
are capable of directing the expression of genes to which they are
operatively-linked. Such vectors are referred to herein as
"expression vectors." Common expression vectors of utility in
recombinant DNA techniques are often in the form of plasmids.
[0555] Recombinant expression vectors can comprise a nucleic acid
of the invention in a form suitable for expression of the nucleic
acid in a host cell, which means that the recombinant expression
vectors include one or more regulatory elements, which may be
selected on the basis of the host cells to be used for expression,
that is operatively-linked to the nucleic acid sequence to be
expressed. Within a recombinant expression vector, "operably
linked" is intended to mean that the nucleotide sequence of
interest is linked to the regulatory element(s) in a manner that
allows for expression of the nucleotide sequence (e.g., in an in
vitro transcription/translation system or in a host cell when the
vector is introduced into the host cell). With regards to
recombination and cloning methods, mention is made of U.S. patent
application Ser. No. 10/815,730, published Sep. 2, 2004 as US
2004-0171156 A1, the contents of which are herein incorporated by
reference in their entirety.
[0556] Aspects of the invention relate to bicistronic vectors for
chimeric RNA and Cas9. Bicistronic expression vectors for chimeric
RNA and Cas9 are preferred. In general and particularly in this
embodiment Cas9 is preferably driven by the CBh promoter. The
chimeric RNA may preferably be driven by a Pol III promoter, such
as a U6 promoter. Ideally the two are combined. The chimeric guide
RNA typically comprises, consists essentially of, or consists of a
20 bp guide sequence (Ns) and this may be joined to the tracr
sequence (running from the first "U" of the lower strand to the end
of the transcript). The tracr sequence may be truncated at various
positions as indicated. The guide and tracr sequences are separated
by the tracr-mate sequence, which may be GUUUUAGAGCUA (SEQ ID NO:
48). This may be followed by the loop sequence GAAA as shown. Both
of these are preferred examples. Applicants have demonstrated
Cas9-mediated indels at the human EMX1 and PVALB loci by SURVEYOR
assays. ChiRNAs are indicated by their "+n" designation, and crRNA
refers to a hybrid RNA where guide and tracr sequences are
expressed as separate transcripts. Throughout this application,
chimeric RNA may also be called single guide, or synthetic guide
RNA (sgRNA).
[0557] In some embodiments, a loop in the guide RNA is provided.
This may be a stem loop or a tetra loop. The loop is preferably
GAAA, but it is not limited to this sequence or indeed to being
only 4 bp in length. Indeed, preferred loop forming sequences for
use in hairpin structures are four nucleotides in length, and most
preferably have the sequence GAAA. However, longer or shorter loop
sequences may be used, as may alternative sequences. The sequences
preferably include a nucleotide triplet (for example, AAA), and an
additional nucleotide (for example C or G). Examples of loop
forming sequences include CAAA and AAAG. In practicing any of the
methods disclosed herein, a suitable vector can be introduced to a
cell or an embryo via one or more methods known in the art,
including without limitation, microinjection, electroporation,
sonoporation, biolistics, calcium phosphate-mediated transfection,
cationic transfection, liposome transfection, dendrimer
transfection, heat shock transfection, nucleofection transfection,
magnetofection, lipofection, impalefection, optical transfection,
proprietary agent-enhanced uptake of nucleic acids, and delivery
via liposomes, immunoliposomes, virosomes, or artificial virions.
In some methods, the vector is introduced into an embryo by
microinjection. The vector or vectors may be microinjected into the
nucleus or the cytoplasm of the embryo. In some methods, the vector
or vectors may be introduced into a cell by nucleofection.
[0558] The term "regulatory element" is intended to include
promoters, enhancers, internal ribosomal entry sites (IRES), and
other expression control elements (e.g., transcription termination
signals, such as polyadenylation signals and poly-U sequences).
Such regulatory elements are described, for example, in Goeddel,
GENE EXPRESSION TECHNOLOGY: METHODS IN ENZYMOLOGY 185, Academic
Press, San Diego, Calif. (1990). Regulatory elements include those
that direct constitutive expression of a nucleotide sequence in
many types of host cell and those that direct expression of the
nucleotide sequence only in certain host cells (e.g.,
tissue-specific regulatory sequences). A tissue-specific promoter
may direct expression primarily in a desired tissue of interest,
such as muscle, neuron, bone, skin, blood, specific organs (e.g.,
liver, pancreas), or particular cell types (e.g., lymphocytes).
Regulatory elements may also direct expression in a
temporal-dependent manner, such as in a cell-cycle dependent or
developmental stage-dependent manner, which may or may not also be
tissue or cell-type specific. In some embodiments, a vector
comprises one or more pol III promoter (e.g., 1, 2, 3, 4, 5, or
more pol III promoters), one or more pol II promoters (e.g., 1, 2,
3, 4, 5, or more pol II promoters), one or more pol I promoters
(e.g., 1, 2, 3, 4, 5, or more pol I promoters), or combinations
thereof. Examples of pol III promoters include, but are not limited
to, U6 and H1 promoters. Examples of pol II promoters include, but
are not limited to, the retroviral Rous sarcoma virus (RSV) LTR
promoter (optionally with the RSV enhancer), the cytomegalovirus
(CMV) promoter (optionally with the CMV enhancer) [see, e.g.,
Boshart et al, Cell, 41:521-530 (1985)], the SV40 promoter, the
dihydrofolate reductase promoter, the .beta.-actin promoter, the
phosphoglycerol kinase (PGK) promoter, and the EF1.alpha. promoter.
Also encompassed by the term "regulatory element" are enhancer
elements, such as WPRE; CMV enhancers; the R-U5' segment in LTR of
HTLV-I (Mol. Cell. Biol., Vol. 8(1), p. 466-472, 1988); SV40
enhancer; and the intron sequence between exons 2 and 3 of rabbit
.beta.-globin (Proc. Natl. Acad. Sci. USA., Vol. 78(3), p. 1527-31,
1981). It will be appreciated by those skilled in the art that the
design of the expression vector can depend on such factors as the
choice of the host cell to be transformed, the level of expression
desired, etc. A vector can be introduced into host cells to thereby
produce transcripts, proteins, or peptides, including fusion
proteins or peptides, encoded by nucleic acids as described herein
(e.g., clustered regularly interspersed short palindromic repeats
(CRISPR) transcripts, proteins, enzymes, mutant forms thereof,
fusion proteins thereof, etc.). With regards to regulatory
sequences, mention is made of U.S. patent application Ser. No.
10/491,026, the contents of which are incorporated by reference
herein in their entirety. With regards to promoters, mention is
made of PCT publication WO 2011/028929 and U.S. application Ser.
No. 12/511,940, the contents of which are incorporated by reference
herein in their entirety.
[0559] Vectors can be designed for expression of CRISPR transcripts
(e.g., nucleic acid transcripts, proteins, or enzymes) in
prokaryotic or eukaryotic cells. For example, CRISPR transcripts
can be expressed in bacterial cells such as Escherichia coli,
insect cells (using baculovirus expression vectors), yeast cells,
or mammalian cells. Suitable host cells are discussed further in
Goeddel, GENE EXPRESSION TECHNOLOGY: METHODS IN ENZYMOLOGY 185,
Academic Press, San Diego, Calif. (1990). Alternatively, the
recombinant expression vector can be transcribed and translated in
vitro, for example using T7 promoter regulatory sequences and T7
polymerase.
[0560] Vectors may be introduced and propagated in a prokaryote or
prokaryotic cell. In some embodiments, a prokaryote is used to
amplify copies of a vector to be introduced into a eukaryotic cell
or as an intermediate vector in the production of a vector to be
introduced into a eukaryotic cell (e.g., amplifying a plasmid as
part of a viral vector packaging system). In some embodiments, a
prokaryote is used to amplify copies of a vector and express one or
more nucleic acids, such as to provide a source of one or more
proteins for delivery to a host cell or host organism. Expression
of proteins in prokaryotes is most often carried out in Escherichia
coli with vectors containing constitutive or inducible promoters
directing the expression of either fusion or non-fusion proteins.
Fusion vectors add a number of amino acids to a protein encoded
therein, such as to the amino terminus of the recombinant protein.
Such fusion vectors may serve one or more purposes, such as: (i) to
increase expression of recombinant protein; (ii) to increase the
solubility of the recombinant protein; and (iii) to aid in the
purification of the recombinant protein by acting as a ligand in
affinity purification. Often, in fusion expression vectors, a
proteolytic cleavage site is introduced at the junction of the
fusion moiety and the recombinant protein to enable separation of
the recombinant protein from the fusion moiety subsequent to
purification of the fusion protein. Such enzymes, and their cognate
recognition sequences, include Factor Xa, thrombin and
enterokinase. Example fusion expression vectors include pGEX
(Pharmacia Biotech Inc; Smith and Johnson, 1988. Gene 67: 31-40),
pMAL (New England Biolabs, Beverly, Mass.) and pRIT5 (Pharmacia,
Piscataway, N.J.) that fuse glutathione S-transferase (GST),
maltose E binding protein, or protein A, respectively, to the
target recombinant protein.
[0561] Examples of suitable inducible non-fusion E. coli expression
vectors include pTrc (Amrann et al., (1988) Gene 69:301-315) and
pET 11d (Studier et al., GENE EXPRESSION TECHNOLOGY: METHODS IN
ENZYMOLOGY 185, Academic Press, San Diego, Calif. (1990)
60-89).
[0562] In some embodiments, a vector is a yeast expression vector.
Examples of vectors for expression in yeast Saccharomyces cerivisae
include pYepSec1 (Baldari, et al., 1987. EMBO J. 6: 229-234), pMFa
(Kuijan and Herskowitz, 1982. Cell 30: 933-943), pJRY88 (Schultz et
al., 1987. Gene 54: 113-123), pYES2 (Invitrogen Corporation, San
Diego, Calif.), and picZ (InVitrogen Corp, San Diego, Calif.).
[0563] In some embodiments, a vector drives protein expression in
insect cells using baculovirus expression vectors. Baculovirus
vectors available for expression of proteins in cultured insect
cells (e.g., SF9 cells) include the pAc series (Smith, et al.,
1983. Mol. Cell. Biol. 3: 2156-2165) and the pVL series (Lucklow
and Summers, 1989. Virology 170: 31-39).
[0564] In some embodiments, a vector is capable of driving
expression of one or more sequences in mammalian cells using a
mammalian expression vector. Examples of mammalian expression
vectors include pCDM8 (Seed, 1987. Nature 329: 840) and pMT2PC
(Kaufman, et al., 1987. EMBO J. 6: 187-195). When used in mammalian
cells, the expression vector's control functions are typically
provided by one or more regulatory elements. For example, commonly
used promoters are derived from polyoma, adenovirus 2,
cytomegalovirus, simian virus 40, and others disclosed herein and
known in the art. For other suitable expression systems for both
prokaryotic and eukaryotic cells see, e.g., Chapters 16 and 17 of
Sambrook, et al., MOLECULAR CLONING: A LABORATORY MANUAL. 2nd ed.,
Cold Spring Harbor Laboratory, Cold Spring Harbor Laboratory Press,
Cold Spring Harbor, N.Y., 1989.
[0565] In some embodiments, the recombinant mammalian expression
vector is capable of directing expression of the nucleic acid
preferentially in a particular cell type (e.g., tissue-specific
regulatory elements are used to express the nucleic acid).
Tissue-specific regulatory elements are known in the art.
Non-limiting examples of suitable tissue-specific promoters include
the albumin promoter (liver-specific; Pinkert, et al., 1987. Genes
Dev. 1: 268-277), lymphoid-specific promoters (Calame and Eaton,
1988. Adv. Immunol. 43: 235-275), in particular promoters of T cell
receptors (Winoto and Baltimore, 1989. EMBO J. 8: 729-733) and
immunoglobulins (Baneiji, et al., 1983. Cell 33: 729-740; Queen and
Baltimore, 1983. Cell 33: 741-748), neuron-specific promoters
(e.g., the neurofilament promoter; Byrne and Ruddle, 1989. Proc.
Natl. Acad. Sci. USA 86: 5473-5477), pancreas-specific promoters
(Edlund, et al., 1985. Science 230: 912-916), and mammary
gland-specific promoters (e.g., milk whey promoter; U.S. Pat. No.
4,873,316 and European Application Publication No. 264,166).
Developmentally-regulated promoters are also encompassed, e.g., the
murine hox promoters (Kessel and Gruss, 1990. Science 249: 374-379)
and the .alpha.-fetoprotein promoter (Campes and Tilghman, 1989.
Genes Dev. 3: 537-546). With regards to these prokaryotic and
eukaryotic vectors, mention is made of U.S. Pat. No. 6,750,059, the
contents of which are incorporated by reference herein in their
entirety. Other embodiments of the invention may relate to the use
of viral vectors, with regards to which mention is made of U.S.
patent application Ser. No. 13/092,085, the contents of which are
incorporated by reference herein in their entirety. Tissue-specific
regulatory elements are known in the art and in this regard,
mention is made of U.S. Pat. No. 7,776,321, the contents of which
are incorporated by reference herein in their entirety.
[0566] In some embodiments, a regulatory element is operably linked
to one or more elements of a CRISPR system so as to drive
expression of the one or more elements of the CRISPR system. In
general, CRISPRs (Clustered Regularly Interspaced Short Palindromic
Repeats), also known as SPIDRs (SPacer Interspersed Direct
Repeats), constitute a family of DNA loci that are usually specific
to a particular bacterial species. The CRISPR locus comprises a
distinct class of interspersed short sequence repeats (SSRs) that
were recognized in E. coli (Ishino et al., J. Bacteriol.,
169:5429-5433 [1987]; and Nakata et al., J. Bacteriol.,
171:3553-3556 [1989]), and associated genes. Similar interspersed
SSRs have been identified in Haloferax mediterranei, Streptococcus
pyogenes, Anabaena, and Mycobacterium tuberculosis (See, Groenen et
al., Mol. Microbiol., 10:1057-1065 [1993]; Hoe et al., Emerg.
Infect. Dis., 5:254-263 [1999]; Masepohl et al., Biochim. Biophys.
Acta 1307:26-30 [1996]; and Mojica et al., Mol. Microbiol.,
17:85-93 [1995]). The CRISPR loci typically differ from other SSRs
by the structure of the repeats, which have been termed short
regularly spaced repeats (SRSRs) (Janssen et al., OMICS J. Integ.
Biol., 6:23-33 [2002]; and Mojica et al., Mol. Microbiol.,
36:244-246 [2000]). In general, the repeats are short elements that
occur in clusters that are regularly spaced by unique intervening
sequences with a substantially constant length (Mojica et al.,
[2000], supra). Although the repeat sequences are highly conserved
between strains, the number of interspersed repeats and the
sequences of the spacer regions typically differ from strain to
strain (van Embden et al., J. Bacteriol., 182:2393-2401 [2000]).
CRISPR loci have been identified in more than 40 prokaryotes (See
e.g., Jansen et al., Mol. Microbiol., 43:1565-1575 [2002]; and
Mojica et al., [2005]) including, but not limited to Aeropyrum,
Pyrobaculum, Sulfolobus, Archaeoglobus, Halocarcula,
Methanobacterium, Methanococcus, Methanosarcina, Methanopyrus,
Pyrococcus, Picrophilus, Thermoplasma, Corynebacterium,
Mycobacterium, Streptomyces, Aquifex, Porphyromonas, Chlorobium,
Thermus, Bacillus, Listeria, Staphylococcus, Clostridium,
Thermoanaerobacter, Mycoplasma, Fusobacterium, Azarcus,
Chromobacterium, Neisseria, Nitrosomonas, Desulfovibrio, Geobacter,
Myxococcus, Campylobacter, Wolinella, Acinetobacter, Envinia,
Escherichia, Legionella, Methylococcus, Pasteurella,
Photobacterium, Salmonella, Xanthomonas, Yersinia, Treponema, and
Thermotoga.
[0567] In general, "CRISPR system" refers collectively to
transcripts and other elements involved in the expression of or
directing the activity of CRISPR-associated ("Cas") genes,
including sequences encoding a Cas gene, a tracr (trans-activating
CRISPR) sequence (e.g. tracrRNA or an active partial tracrRNA), a
tracr-mate sequence (encompassing a "direct repeat" and a
tracrRNA-processed partial direct repeat in the context of an
endogenous CRISPR system), a guide sequence (also referred to as a
"spacer" in the context of an endogenous CRISPR system), or other
sequences and transcripts from a CRISPR locus. In some embodiments,
one or more elements of a CRISPR system is derived from a type I,
type II, or type III CRISPR system. In some embodiments, one or
more elements of a CRISPR system is derived from a particular
organism comprising an endogenous CRISPR system, such as
Streptococcus pyogenes. In general, a CRISPR system is
characterized by elements that promote the formation of a CRISPR
complex at the site of a target sequence (also referred to as a
protospacer in the context of an endogenous CRISPR system). In the
context of formation of a CRISPR complex, "target sequence" refers
to a sequence to which a guide sequence is designed to have
complementarity, where hybridization between a target sequence and
a guide sequence promotes the formation of a CRISPR complex. Full
complementarity is not necessarily required, provided there is
sufficient complementarity to cause hybridization and promote
formation of a CRISPR complex. A target sequence may comprise any
polynucleotide, such as DNA or RNA polynucleotides. In some
embodiments, a target sequence is located in the nucleus or
cytoplasm of a cell. In some embodiments, the target sequence may
be within an organelle of a eukaryotic cell, for example,
mitochondrion or chloroplast. A sequence or template that may be
used for recombination into the targeted locus comprising the
target sequences is referred to as an "editing template" or
"editing polynucleotide" or "editing sequence". In aspects of the
invention, an exogenous template polynucleotide may be referred to
as an editing template. In an aspect of the invention the
recombination is homologous recombination.
[0568] Typically, in the context of an endogenous CRISPR system,
formation of a CRISPR complex (comprising a guide sequence
hybridized to a target sequence and complexed with one or more Cas
proteins) results in cleavage of one or both strands in or near
(e.g. within 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 50, or more base
pairs from) the target sequence. Without wishing to be bound by
theory, the tracr sequence, which may comprise or consist of all or
a portion of a wild-type tracr sequence (e.g. about or more than
about 20, 26, 32, 45, 48, 54, 63, 67, 85, or more nucleotides of a
wild-type tracr sequence), may also form part of a CRISPR complex,
such as by hybridization along at least a portion of the tracr
sequence to all or a portion of a tracr mate sequence that is
operably linked to the guide sequence. In some embodiments, the
tracr sequence has sufficient complementarity to a tracr mate
sequence to hybridize and participate in formation of a CRISPR
complex. As with the target sequence, it is believed that complete
complementarity is not needed, provided there is sufficient to be
functional. In some embodiments, the tracr sequence has at least
50%, 60%, 70%, 80%, 90%, 95% or 99% of sequence complementarity
along the length of the tracr mate sequence when optimally aligned.
In some embodiments, one or more vectors driving expression of one
or more elements of a CRISPR system are introduced into a host cell
such that expression of the elements of the CRISPR system direct
formation of a CRISPR complex at one or more target sites. For
example, a Cas9 enzyme, a guide sequence linked to a tracr-mate
sequence, and a tracr sequence could each be operably linked to
separate regulatory elements on separate vectors. Alternatively,
two or more of the elements expressed from the same or different
regulatory elements, may be combined in a single vector, with one
or more additional vectors providing any components of the CRISPR
system not included in the first vector. CRISPR system elements
that are combined in a single vector may be arranged in any
suitable orientation, such as one element located 5' with respect
to ("upstream" of) or 3' with respect to ("downstream" of) a second
element. The coding sequence of one element may be located on the
same or opposite strand of the coding sequence of a second element,
and oriented in the same or opposite direction. In some
embodiments, a single promoter drives expression of a transcript
encoding a CRISPR enzyme and one or more of the guide sequence,
tracr mate sequence (optionally operably linked to the guide
sequence), and a tracr sequence embedded within one or more intron
sequences (e.g. each in a different intron, two or more in at least
one intron, or all in a single intron). In some embodiments, the
CRISPR enzyme, guide sequence, tracr mate sequence, and tracr
sequence are operably linked to and expressed from the same
promoter.
[0569] In some embodiments, a vector comprises one or more
insertion sites, such as a restriction endonuclease recognition
sequence (also referred to as a "cloning site"). In some
embodiments, one or more insertion sites (e.g. about or more than
about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more insertion sites) are
located upstream and/or downstream of one or more sequence elements
of one or more vectors. In some embodiments, a vector comprises an
insertion site upstream of a tracr mate sequence, and optionally
downstream of a regulatory element operably linked to the tracr
mate sequence, such that following insertion of a guide sequence
into the insertion site and upon expression the guide sequence
directs sequence-specific binding of a CRISPR complex to a target
sequence in a eukaryotic cell. In some embodiments, a vector
comprises two or more insertion sites, each insertion site being
located between two tracr mate sequences so as to allow insertion
of a guide sequence at each site. In such an arrangement, the two
or more guide sequences may comprise two or more copies of a single
guide sequence, two or more different guide sequences, or
combinations of these. When multiple different guide sequences are
used, a single expression construct may be used to target CRISPR
activity to multiple different, corresponding target sequences
within a cell. For example, a single vector may comprise about or
more than about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, or more
guide sequences. In some embodiments, about or more than about 1,
2, 3, 4, 5, 6, 7, 8, 9, 10, or more such guide-sequence-containing
vectors may be provided, and optionally delivered to a cell.
[0570] In some embodiments, a vector comprises a regulatory element
operably linked to an enzyme-coding sequence encoding a CRISPR Cas9
protein. These enzymes are known; for example, the amino acid
sequence of S. pyogenes Cas9 protein may be found in the SwissProt
database under accession number Q99ZW2. In some embodiments, the
unmodified CRISPR enzyme has DNA cleavage activity, such as Cas9.
The CRISPR enzyme is Cas9, and may be Cas9 from S. pyogenes or S.
pneumoniae. In some embodiments, the CRISPR enzyme directs cleavage
of one or both strands at the location of a target sequence, such
as within the target sequence and/or within the complement of the
target sequence. In some embodiments, the CRISPR enzyme directs
cleavage of one or both strands within about 1, 2, 3, 4, 5, 6, 7,
8, 9, 10, 15, 20, 25, 50, 100, 200, 500, or more base pairs from
the first or last nucleotide of a target sequence. In some
embodiments, a vector encodes a CRISPR enzyme that is mutated to
with respect to a corresponding wild-type enzyme such that the
mutated CRISPR enzyme lacks the ability to cleave one or both
strands of a target polynucleotide containing a target sequence.
For example, an aspartate-to-alanine substitution (D10A) in the
RuvC I catalytic domain of Cas9 from S. pyogenes converts Cas9 from
a nuclease that cleaves both strands to a nickase (cleaves a single
strand). Other examples of mutations that render Cas9 a nickase
include, without limitation, H840A, N854A, and N863A. In some
embodiments, a Cas9 nickase may be used in combination with guide
sequenc(es), e.g., two guide sequences, which target respectively
sense and antisense strands of the DNA target. This combination
allows both strands to be nicked and used to induce NHEJ.
Applicants have demonstrated (data not shown) the efficacy of two
nickase targets (i.e., sgRNAs targeted at the same location but to
different strands of DNA) in inducing mutagenic NHEJ. A single
nickase (Cas9-D10A with a single sgRNA) is unable to induce NHEJ
and create indels but Applicants have shown that double nickase
(Cas9-D10A and two sgRNAs targeted to different strands at the same
location) can do so in human embryonic stem cells (hESCs). The
efficiency is about 50% of nuclease (i.e., regular Cas9 without D10
mutation) in hESCs.
[0571] As a further example, two or more catalytic domains of Cas9
(RuvC I, RuvC II, and RuvC III) may be mutated to produce a mutated
Cas9 substantially lacking all DNA cleavage activity. In some
embodiments, a D10A mutation is combined with one or more of H840A,
N854A, or N863A mutations to produce a Cas9 enzyme substantially
lacking all DNA cleavage activity. In some embodiments, a CRISPR
enzyme is considered to substantially lack all DNA cleavage
activity when the DNA cleavage activity of the mutated enzyme is
less than about 25%, 10%, 5%, 1%, 0.1%, 0.01%, or lower with
respect to its non-mutated form. Other mutations may be useful;
where the Cas9 or other CRISPR enzyme is from a species other than
S. pyogenes, mutations in corresponding amino acids may be made to
achieve similar effects.
[0572] In some embodiments, a coding sequence encoding a CRISPR
enzyme is codon optimized for expression in particular cells, such
as eukaryotic cells. The eukaryotic cells may be those of or
derived from a particular organism, such as a mammal, including but
not limited to human, mouse, rat, rabbit, dog, or non-human
primate. In general, codon optimization refers to a process of
modifying a nucleic acid sequence for enhanced expression in the
host cells of interest by replacing at least one codon (e.g. about
or more than about 1, 2, 3, 4, 5, 10, 15, 20, 25, 50, or more
codons) of the native sequence with codons that are more frequently
or most frequently used in the genes of that host cell while
maintaining the native amino acid sequence. Various species exhibit
particular bias for certain codons of a particular amino acid.
Codon bias (differences in codon usage between organisms) often
correlates with the efficiency of translation of messenger RNA
(mRNA), which is in turn believed to be dependent on, among other
things, the properties of the codons being translated and the
availability of particular transfer RNA (tRNA) molecules. The
predominance of selected tRNAs in a cell is generally a reflection
of the codons used most frequently in peptide synthesis.
Accordingly, genes can be tailored for optimal gene expression in a
given organism based on codon optimization. Codon usage tables are
readily available, for example, at the "Codon Usage Database", and
these tables can be adapted in a number of ways. See Nakamura, Y.,
et al. "Codon usage tabulated from the international DNA sequence
databases: status for the year 2000" Nucl. Acids Res. 28:292
(2000). Computer algorithms for codon optimizing a particular
sequence for expression in a particular host cell are also
available, such as Gene Forge (Aptagen; Jacobus, Pa.), are also
available. In some embodiments, one or more codons (e.g. 1, 2, 3,
4, 5, 10, 15, 20, 25, 50, or more, or all codons) in a sequence
encoding a CRISPR enzyme correspond to the most frequently used
codon for a particular amino acid.
[0573] In some embodiments, a vector encodes a CRISPR enzyme
comprising one or more nuclear localization sequences (NLSs), such
as about or more than about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more
NLSs. In some embodiments, the CRISPR enzyme comprises about or
more than about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more NLSs at or
near the amino-terminus, about or more than about 1, 2, 3, 4, 5, 6,
7, 8, 9, 10, or more NLSs at or near the carboxy-terminus, or a
combination of these (e.g. one or more NLS at the amino-terminus
and one or more NLS at the carboxy terminus). When more than one
NLS is present, each may be selected independently of the others,
such that a single NLS may be present in more than one copy and/or
in combination with one or more other NLSs present in one or more
copies. In a preferred embodiment of the invention, the CRISPR
enzyme comprises at most 6 NLSs. In some embodiments, an NLS is
considered near the N- or C-terminus when the nearest amino acid of
the NLS is within about 1, 2, 3, 4, 5, 10, 15, 20, 25, 30, 40, 50,
or more amino acids along the polypeptide chain from the N- or
C-terminus. Typically, an NLS consists of one or more short
sequences of positively charged lysines or arginines exposed on the
protein surface, but other types of NLS are known. Non-limiting
examples of NLSs include an NLS sequence derived from: the NLS of
the SV40 virus large T-antigen, having the amino acid sequence
PKKKRKV (SEQ ID NO: 1); the NLS from nucleoplasmin (e.g. the
nucleoplasmin bipartite NLS with the sequence KRPAATKKAGQAKKKK (SEQ
ID NO: 2)); the c-myc NLS having the amino acid sequence PAAKRVKLD
(SEQ ID NO: 3) or RQRRNELKRSP (SEQ ID NO: 4); the hRNPA1 M9 NLS
having the sequence NQSSNFGPMKGGNFGGRSSGPYGGGGQYFAKPRNQGGY (SEQ ID
NO: 5); the sequence RMRIZFKNKGKDTAELRRRRVEVSVELRKAKKDEQILKRRNV
(SEQ ID NO: 6) of the IBB domain from importin-alpha; the sequences
VSRKRPRP (SEQ ID NO: 7) and PPKKARED (SEQ ID NO: 8) of the myoma T
protein; the sequence PQPKKKPL (SEQ ID NO: 9) of human p53; the
sequence SALIKKKKKMAP (SEQ ID NO: 10) of mouse c-abl IV; the
sequences DRLRR (SEQ ID NO: 11) and PKQKKRK (SEQ ID NO: 12) of the
influenza virus NS1; the sequence RKLKKKIKKL (SEQ ID NO: 13) of the
Hepatitis virus delta antigen; the sequence REKKKFLKRR (SEQ ID NO:
14) of the mouse Mx1 protein; the sequence KRKGDEVDGVDEVAKKKSKK
(SEQ ID NO: 15) of the human poly(ADP-ribose) polymerase; and the
sequence RKCLQAGMNLEARKTKK (SEQ ID NO: 16) of the steroid hormone
receptors (human) glucocorticoid.
[0574] In general, the one or more NLSs are of sufficient strength
to drive accumulation of the CRISPR enzyme in a detectable amount
in the nucleus of a eukaryotic cell. In general, strength of
nuclear localization activity may derive from the number of NLSs in
the CRISPR enzyme, the particular NLS(s) used, or a combination of
these factors. Detection of accumulation in the nucleus may be
performed by any suitable technique. For example, a detectable
marker may be fused to the CRISPR enzyme, such that location within
a cell may be visualized, such as in combination with a means for
detecting the location of the nucleus (e.g. a stain specific for
the nucleus such as DAPI). Examples of detectable markers include
fluorescent proteins (such as Green fluorescent proteins, or GFP;
RFP; CFP), and epitope tags (HA tag, flag tag, SNAP tag). Cell
nuclei may also be isolated from cells, the contents of which may
then be analyzed by any suitable process for detecting protein,
such as immunohistochemistry, Western blot, or enzyme activity
assay. Accumulation in the nucleus may also be determined
indirectly, such as by an assay for the effect of CRISPR complex
formation (e.g. assay for DNA cleavage or mutation at the target
sequence, or assay for altered gene expression activity affected by
CRISPR complex formation and/or CRISPR enzyme activity), as
compared to a control no exposed to the CRISPR enzyme or complex,
or exposed to a CRISPR enzyme lacking the one or more NLSs.
[0575] In general, a guide sequence is any polynucleotide sequence
having sufficient complementarity with a target polynucleotide
sequence to hybridize with the target sequence and direct
sequence-specific binding of a CRISPR complex to the target
sequence. In some embodiments, the degree of complementarity
between a guide sequence and its corresponding target sequence,
when optimally aligned using a suitable alignment algorithm, is
about or more than about 50%, 60%, 75%, 80%, 85%, 90%, 95%, 97.5%,
99%, or more. Optimal alignment may be determined with the use of
any suitable algorithm for aligning sequences, non-limiting example
of which include the Smith-Waterman algorithm, the Needleman-Wunsch
algorithm, algorithms based on the Burrows-Wheeler Transform (e.g.
the Burrows Wheeler Aligner), ClustalW, Clustal X, BLAT, Novoalign
(Novocraft Technologies, ELAND (Illumina, San Diego, Calif.), SOAP
(available at soap.genomics.org.cn), and Maq (available at
maq.sourceforge.net). In some embodiments, a guide sequence is
about or more than about 5, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19,
20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 35, 40, 45, 50, 75, or
more nucleotides in length. In some embodiments, a guide sequence
is less than about 75, 50, 45, 40, 35, 30, 25, 20, 15, 12, or fewer
nucleotides in length. The ability of a guide sequence to direct
sequence-specific binding of a CRISPR complex to a target sequence
may be assessed by any suitable assay. For example, the components
of a CRISPR system sufficient to form a CRISPR complex, including
the guide sequence to be tested, may be provided to a host cell
having the corresponding target sequence, such as by transfection
with vectors encoding the components of the CRISPR sequence,
followed by an assessment of preferential cleavage within the
target sequence, such as by Surveyor assay as described herein.
Similarly, cleavage of a target polynucleotide sequence may be
evaluated in a test tube by providing the target sequence,
components of a CRISPR complex, including the guide sequence to be
tested and a control guide sequence different from the test guide
sequence, and comparing binding or rate of cleavage at the target
sequence between the test and control guide sequence reactions.
Other assays are possible, and will occur to those skilled in the
art.
[0576] A guide sequence may be selected to target any target
sequence. In some embodiments, the target sequence is a sequence
within a genome of a cell. Exemplary target sequences include those
that are unique in the target genome. For example, for the S.
pyogenes Cas9, a unique target sequence in a genome may include a
Cas9 target site of the form MMMMMMMMNNNNNNNNNNNNXGG (SEQ ID NO:
62) where NNNNNNNNNNNNXGG (SEQ ID NO: 63) (N is A, G, T, or C; and
X can be anything) has a single occurrence in the genome. A unique
target sequence in a genome may include an S. pyogenes Cas9 target
site of the form MMMMMMMMMNNNNNNNNNNNXGG (SEQ ID NO: 64) where
NNNNNNNNNNNXGG (SEQ ID NO: 65) (N is A, G, T, or C; and X can be
anything) has a single occurrence in the genome. For the S.
thermophilus CRISPR1 Cas9, a unique target sequence in a genome may
include a Cas9 target site of the form MMMMMMMMNNNNNNNNNNNNXXAGAAW
(SEQ ID NO: 17) where NNNNNNNNNNNNXXAGAAW (SEQ ID NO: 18) (N is A,
G, T, or C; X can be anything; and W is A or T) has a single
occurrence in the genome. A unique target sequence in a genome may
include an S. thermophilus CRISPR1 Cas9 target site of the form
MMMMMMMMMNNNNNNNNNNNXXAGAAW (SEQ ID NO: 19) where
NNNNNNNNNNNXXAGAAW (SEQ ID NO: 20) (N is A, G, T, or C; X can be
anything; and W is A or T) has a single occurrence in the genome.
For the S. pyogenes Cas9, a unique target sequence in a genome may
include a Cas9 target site of the form MMMMMMMMNNNNNNNNNNNNXGGXG
(SEQ ID NO: 66) where NNNNNNNNNNNNXGGXG (SEQ ID NO: 67) (N is A, G,
T, or C; and X can be anything) has a single occurrence in the
genome. A unique target sequence in a genome may include an S.
pyogenes Cas9 target site of the form MMMMMMMMMNNNNNNNNNNNXGGXG
(SEQ ID NO: 68) where NNNNNNNNNNNXGGXG (SEQ ID NO: 69) (N is A, G,
T, or C; and X can be anything) has a single occurrence in the
genome. In each of these sequences "M" may be A, G, T, or C, and
need not be considered in identifying a sequence as unique.
[0577] In some embodiments, a guide sequence is selected to reduce
the degree of secondary structure within the guide sequence.
Secondary structure may be determined by any suitable
polynucleotide folding algorithm. Some programs are based on
calculating the minimal Gibbs free energy. An example of one such
algorithm is mFold, as described by Zuker and Stiegler (Nucleic
Acids Res. 9 (1981), 133-148). Another example folding algorithm is
the online webserver RNAfold, developed at Institute for
Theoretical Chemistry at the University of Vienna, using the
centroid structure prediction algorithm (see e.g. A. R. Gruber et
al., 2008, Cell 106(1): 23-24; and PA Carr and GM Church, 2009,
Nature Biotechnology 27(12): 1151-62). Further algorithms may be
found in U.S. application Ser. No. ______ (attorney docket
44790.11.2022; Broad Reference BI-2013/004A); incorporated herein
by reference.
[0578] In general, a tracr mate sequence includes any sequence that
has sufficient complementarity with a tracr sequence to promote one
or more of: (1) excision of a guide sequence flanked by tracr mate
sequences in a cell containing the corresponding tracr sequence;
and (2) formation of a CRISPR complex at a target sequence, wherein
the CRISPR complex comprises the tracr mate sequence hybridized to
the tracr sequence. In general, degree of complementarity is with
reference to the optimal alignment of the tracr mate sequence and
tracr sequence, along the length of the shorter of the two
sequences. Optimal alignment may be determined by any suitable
alignment algorithm, and may further account for secondary
structures, such as self-complementarity within either the tracr
sequence or tracr mate sequence. In some embodiments, the degree of
complementarity between the tracr sequence and tracr mate sequence
along the length of the shorter of the two when optimally aligned
is about or more than about 25%, 30%, 40%, 50%, 60%, 70%, 80%, 90%,
95%, 97.5%, 99%, or higher. ExIn some embodiments, the tracr
sequence is about or more than about 5, 6, 7, 8, 9, 10, 11, 12, 13,
14, 15, 16, 17, 18, 19, 20, 25, 30, 40, 50, or more nucleotides in
length. In some embodiments, the tracr sequence and tracr mate
sequence are contained within a single transcript, such that
hybridization between the two produces a transcript having a
secondary structure, such as a hairpin. Preferred loop forming
sequences for use in hairpin structures are four nucleotides in
length, and most preferably have the sequence GAAA. However, longer
or shorter loop sequences may be used, as may alternative
sequences. The sequences preferably include a nucleotide triplet
(for example, AAA), and an additional nucleotide (for example C or
G). Examples of loop forming sequences include CAAA and AAAG. In an
embodiment of the invention, the transcript or transcribed
polynucleotide sequence has at least two or more hairpins. In
preferred embodiments, the transcript has two, three, four or five
hairpins. In a further embodiment of the invention, the transcript
has at most five hairpins. In some embodiments, the single
transcript further includes a transcription termination sequence;
preferably this is a polyT sequence, for example six T nucleotides.
An example of such a hairpin structure is where the portion of the
sequence 5' of the final "N" and upstream of the loop corresponds
to the tracr mate sequence, and the portion of the sequence 3' of
the loop corresponds to the tracr sequence. Further non-limiting
examples of single polynucleotides comprising a guide sequence, a
tracr mate sequence, and a tracr sequence are as follows (listed 5'
to 3'), where "N" represents a base of a guide sequence, the first
block of lower case letters represent the tracr mate sequence, and
the second block of lower case letters represent the tracr
sequence, and the final poly-T sequence represents the
transcription terminator: (1)
NNNNNNNNNNNNNNNNNNNNgtttttgtactctcaagatttaGAAAtaaatcttgcagaagctacaaagataa-
ggctt catgccgaaatcaacaccctgtcattttatggcagggtgttttcgttatttaaTTTTTT
(SEQ ID NO: 21); (2)
NNNNNNNNNNNNNNNNNNNNgtttttgtactctcaGAAAtgcagaagctacaaagataaggcttcatgccgaa-
atca acaccctgtcattttatggcagggtgttttcgttatttaaTTTTTT (SEQ ID NO:
22); (3)
NNNNNNNNNNNNNNNNNNNNgtttttgtactctcaGAAAtgcagaagctacaaagataaggcttcatgccgaa-
atca acaccctgtcattttatggcagggtgtTTTTTT (SEQ ID NO: 23); (4)
NNNNNNNNNNNNNNNNNNNNgttttagagctaGAAAtagcaagttaaaataaggctagtccgttatcaacttg-
aaaa agtggcaccgagtcggtgcTTTTTT (SEQ ID NO: 24); (5)
NNNNNNNNNNNNNNNNNNNNgttttagagctaGAAATAGcaagttaaaataaggctagtccgttatcaacttg-
aa aaagtgTTTTTTT (SEQ ID NO: 25); and (6)
NNNNNNNNNNNNNNNNNNNNgttttagagctagAAATAGcaagttaaaataaggctagtccgttatcaTTTTT
TTT (SEQ ID NO: 26). In some embodiments, sequences (1) to (3) are
used in combination with Cas9 from S. thermophilus CRISPR1. In some
embodiments, sequences (4) to (6) are used in combination with Cas9
from S. pyogenes. In some embodiments, the tracr sequence is a
separate transcript from a transcript comprising the tracr mate
sequence.
[0579] In some embodiments, a recombination template is also
provided. A recombination template may be a component of another
vector as described herein, contained in a separate vector, or
provided as a separate polynucleotide. In some embodiments, a
recombination template is designed to serve as a template in
homologous recombination, such as within or near a target sequence
nicked or cleaved by a CRISPR enzyme as a part of a CRISPR complex.
A template polynucleotide may be of any suitable length, such as
about or more than about 10, 15, 20, 25, 50, 75, 100, 150, 200,
500, 1000, or more nucleotides in length. In some embodiments, the
template polynucleotide is complementary to a portion of a
polynucleotide comprising the target sequence. When optimally
aligned, a template polynucleotide might overlap with one or more
nucleotides of a target sequences (e.g. about or more than about 1,
5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 60, 70, 80, 90, 100 or more
nucleotides). In some embodiments, when a template sequence and a
polynucleotide comprising a target sequence are optimally aligned,
the nearest nucleotide of the template polynucleotide is within
about 1, 5, 10, 15, 20, 25, 50, 75, 100, 200, 300, 400, 500, 1000,
5000, 10000, or more nucleotides from the target sequence.
[0580] In some embodiments, the CRISPR enzyme is part of a fusion
protein comprising one or more heterologous protein domains (e.g.,
about or more than about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more
domains in addition to the CRISPR enzyme). A CRISPR enzyme fusion
protein may comprise any additional protein sequence, and
optionally a linker sequence between any two domains. Examples of
protein domains that may be fused to a CRISPR enzyme include,
without limitation, epitope tags, reporter gene sequences, and
protein domains having one or more of the following activities:
methylase activity, demethylase activity, transcription activation
activity, transcription repression activity, transcription release
factor activity, histone modification activity, RNA cleavage
activity and nucleic acid binding activity. Non-limiting examples
of epitope tags include histidine (His) tags, V5 tags, FLAG tags,
influenza hemagglutinin (HA) tags, Myc tags, VSV-G tags, and
thioredoxin (Trx) tags. Examples of reporter genes include, but are
not limited to, glutathione-S-transferase (GST), horseradish
peroxidase (HRP), chloramphenicol acetyltransferase (CAT)
beta-galactosidase, beta-glucuronidase, luciferase, green
fluorescent protein (GFP), HcRed, DsRed, cyan fluorescent protein
(CFP), yellow fluorescent protein (YFP), and autofluorescent
proteins including blue fluorescent protein (BFP). A CRISPR enzyme
may be fused to a gene sequence encoding a protein or a fragment of
a protein that bind DNA molecules or bind other cellular molecules,
including but not limited to maltose binding protein (MBP), S-tag,
Lex A DNA binding domain (DBD) fusions, GAL4 DNA binding domain
fusions, and herpes simplex virus (HSV) BP16 protein fusions.
Additional domains that may form part of a fusion protein
comprising a CRISPR enzyme are described in US20110059502,
incorporated herein by reference. In some embodiments, a tagged
CRISPR enzyme is used to identify the location of a target
sequence.
[0581] In some aspects, the invention provides methods comprising
delivering one or more polynucleotides, such as or one or more
vectors as described herein, one or more transcripts thereof,
and/or one or proteins transcribed therefrom, to a host cell. In
some aspects, the invention further provides cells produced by such
methods, and organisms (such as animals, plants, or fungi)
comprising or produced from such cells. In some embodiments, a
CRISPR enzyme in combination with (and optionally complexed with) a
guide sequence is delivered to a cell. Conventional viral and
non-viral based gene transfer methods can be used to introduce
nucleic acids in mammalian cells or target tissues. Such methods
can be used to administer nucleic acids encoding components of a
CRISPR system to cells in culture, or in a host organism. Non-viral
vector delivery systems include DNA plasmids, RNA (e.g. a
transcript of a vector described herein), naked nucleic acid, and
nucleic acid complexed with a delivery vehicle, such as a liposome.
Viral vector delivery systems include DNA and RNA viruses, which
have either episomal or integrated genomes after delivery to the
cell. For a review of gene therapy procedures, see Anderson,
Science 256:808-813 (1992); Nabel & Felgner, TIBTECH 11:211-217
(1993); Mitani & Caskey, TIBTECH 11:162-166 (1993); Dillon,
TIBTECH 11:167-175 (1993); Miller, Nature 357:455-460 (1992); Van
Brunt, Biotechnology 6(10):1149-1154 (1988); Vigne, Restorative
Neurology and Neuroscience 8:35-36 (1995); Kremer &
Perricaudet, British Medical Bulletin 51(1):31-44 (1995); Haddada
et al., in Current Topics in Microbiology and Immunology, Doerfler
and Bohm (eds) (1995); and Yu et al., Gene Therapy 1:13-26
(1994).
[0582] Methods of non-viral delivery of nucleic acids include
lipofection, nucleofection, microinjection, biolistics, virosomes,
liposomes, immunoliposomes, polycation or lipid:nucleic acid
conjugates, naked DNA, artificial virions, and agent-enhanced
uptake of DNA. Lipofection is described in e.g., U.S. Pat. Nos.
5,049,386, 4,946,787; and 4,897,355) and lipofection reagents are
sold commercially (e.g., Transfectam.TM. and Lipofectin.TM.).
Cationic and neutral lipids that are suitable for efficient
receptor-recognition lipofection of polynucleotides include those
of Felgner, WO 91/17424; WO 91/16024. Delivery can be to cells
(e.g. in vitro or ex vivo administration) or target tissues (e.g.
in vivo administration).
[0583] The preparation of lipid:nucleic acid complexes, including
targeted liposomes such as immunolipid complexes, is well known to
one of skill in the art (see, e.g., Crystal, Science 270:404-410
(1995); Blaese et al., Cancer Gene Ther. 2:291-297 (1995); Behr et
al., Bioconjugate Chem. 5:382-389 (1994); Remy et al., Bioconjugate
Chem. 5:647-654 (1994); Gao et al., Gene Therapy 2:710-722 (1995);
Ahmad et al., Cancer Res. 52:4817-4820 (1992); U.S. Pat. Nos.
4,186,183, 4,217,344, 4,235,871, 4,261,975, 4,485,054, 4,501,728,
4,774,085, 4,837,028, and 4,946,787).
[0584] The use of RNA or DNA viral based systems for the delivery
of nucleic acids takes advantage of highly evolved processes for
targeting a virus to specific cells in the body and trafficking the
viral payload to the nucleus. Viral vectors can be administered
directly to patients (in vivo) or they can be used to treat cells
in vitro, and the modified cells may optionally be administered to
patients (ex vivo). Conventional viral based systems could include
retroviral, lentivirus, adenoviral, adeno-associated and herpes
simplex virus vectors for gene transfer. Integration in the host
genome is possible with the retrovirus, lentivirus, and
adeno-associated virus gene transfer methods, often resulting in
long term expression of the inserted transgene. Additionally, high
transduction efficiencies have been observed in many different cell
types and target tissues.
[0585] The tropism of a retrovirus can be altered by incorporating
foreign envelope proteins, expanding the potential target
population of target cells. Lentiviral vectors are retroviral
vectors that are able to transduce or infect non-dividing cells and
typically produce high viral titers. Selection of a retroviral gene
transfer system would therefore depend on the target tissue.
Retroviral vectors are comprised of cis-acting long terminal
repeats with packaging capacity for up to 6-10 kb of foreign
sequence. The minimum cis-acting LTRs are sufficient for
replication and packaging of the vectors, which are then used to
integrate the therapeutic gene into the target cell to provide
permanent transgene expression. Widely used retroviral vectors
include those based upon murine leukemia virus (MuLV), gibbon ape
leukemia virus (GaLV), Simian Immuno deficiency virus (SIV), human
immuno deficiency virus (HIV), and combinations thereof (see, e.g.,
Buchscher et al., J. Virol. 66:2731-2739 (1992); Johann et al., J.
Virol. 66:1635-1640 (1992); Sommnerfelt et al., Virol. 176:58-59
(1990); Wilson et al., J. Virol. 63:2374-2378 (1989); Miller et
al., J. Virol. 65:2220-2224 (1991); PCT/US94/05700). In
applications where transient expression is preferred, adenoviral
based systems may be used. Adenoviral based vectors are capable of
very high transduction efficiency in many cell types and do not
require cell division. With such vectors, high titer and levels of
expression have been obtained. This vector can be produced in large
quantities in a relatively simple system. Adeno-associated virus
("AAV") vectors may also be used to transduce cells with target
nucleic acids, e.g., in the in vitro production of nucleic acids
and peptides, and for in vivo and ex vivo gene therapy procedures
(see, e.g., West et al., Virology 160:38-47 (1987); U.S. Pat. No.
4,797,368; WO 93/24641; Kotin, Human Gene Therapy 5:793-801 (1994);
Muzyczka, J. Clin. Invest. 94:1351 (1994). Construction of
recombinant AAV vectors are described in a number of publications,
including U.S. Pat. No. 5,173,414; Tratschin et al., Mol. Cell.
Biol. 5:3251-3260 (1985); Tratschin, et al., Mol. Cell. Biol.
4:2072-2081 (1984); Hermonat & Muzyczka, PNAS 81:6466-6470
(1984); and Samulski et al., J. Virol. 63:03822-3828 (1989).
[0586] Packaging cells are typically used to form virus particles
that are capable of infecting a host cell. Such cells include 293
cells, which package adenovirus, and .psi.2 cells or PA317 cells,
which package retrovirus. Viral vectors used in gene therapy are
usually generated by producing a cell line that packages a nucleic
acid vector into a viral particle. The vectors typically contain
the minimal viral sequences required for packaging and subsequent
integration into a host, other viral sequences being replaced by an
expression cassette for the polynucleotide(s) to be expressed. The
missing viral functions are typically supplied in trans by the
packaging cell line. For example, AAV vectors used in gene therapy
typically only possess ITR sequences from the AAV genome which are
required for packaging and integration into the host genome. Viral
DNA is packaged in a cell line, which contains a helper plasmid
encoding the other AAV genes, namely rep and cap, but lacking ITR
sequences. The cell line may also be infected with adenovirus as a
helper. The helper virus promotes replication of the AAV vector and
expression of AAV genes from the helper plasmid. The helper plasmid
is not packaged in significant amounts due to a lack of ITR
sequences. Contamination with adenovirus can be reduced by, e.g.,
heat treatment to which adenovirus is more sensitive than AAV.
Additional methods for the delivery of nucleic acids to cells are
known to those skilled in the art. See, for example, US20030087817,
incorporated herein by reference.
[0587] In some embodiments, a host cell is transiently or
non-transiently transfected with one or more vectors described
herein. In some embodiments, a cell is transfected as it naturally
occurs in a subject. In some embodiments, a cell that is
transfected is taken from a subject. In some embodiments, the cell
is derived from cells taken from a subject, such as a cell line. A
wide variety of cell lines for tissue culture are known in the art.
Examples of cell lines include, but are not limited to, C8161,
CCRF-CEM, MOLT, mIMCD-3, NHDF, HeLa-S3, Huh1, Huh4, Huh7, HUVEC,
HASMC, HEKn, HEKa, MiaPaCell, Panc1, PC-3, TF1, CTLL-2, C1R, Rat6,
CV1, RPTE, A10, T24, J82, A375, ARH-77, Calu1, SW480, SW620, SKOV3,
SK-UT, CaCo2, P388D1, SEM-K2, WEHI-231, HB56, TIB55, Jurkat,
J45.01, LRMB, Bcl-1, BC-3, IC21, DLD2, Raw264.7, NRK, NRK-52E,
MRC5, MEF, Hep G2, HeLa B, HeLa T4, COS, COS-1, COS-6, COS-M6A,
BS-C-1 monkey kidney epithelial, BALB/3T3 mouse embryo fibroblast,
3T3 Swiss, 3T3-L1, 132-d5 human fetal fibroblasts; 10.1 mouse
fibroblasts, 293-T, 3T3, 721, 9L, A2780, A2780ADR, A2780cis, A172,
A20, A253, A431, A-549, ALC, B16, B35, BCP-1 cells, BEAS-2B,
bEnd.3, BHK-21, BR 293, BxPC3, C3H-10T1/2, C6/36, Cal-27, CHO,
CHO-7, CHO-IR, CHO-K1, CHO-K2, CHO-T, CHO Dhfr -/-, COR-L23,
COR-L23/CPR, COR-L23/5010, COR-L23/R23, COS-7, COV-434, CML T1,
CMT, CT26, D17, DH82, DU145, DuCaP, EL4, EM2, EM3, EMT6/AR1,
EMT6/AR10.0, FM3, H1299, H69, HB54, HB55, HCA2, HEK-293, HeLa,
Hepa1c1c7, HL-60, HMEC, HT-29, Jurkat, JY cells, K562 cells, Ku812,
KCL22, KG1, KYO1, LNCap, Ma-Mel 1-48, MC-38, MCF-7, MCF-10A,
MDA-MB-231, MDA-MB-468, MDA-MB-435, MDCK II, MDCK II, MOR/0.2R,
MONO-MAC 6, MTD-1A, MyEnd, NCI-H69/CPR, NCI-H69/LX10, NCI-H69/LX20,
NCI-H69/LX4, NIH-3T3, NALM-1, NW-145, OPCN/OPCT cell lines, Peer,
PNT-1A/PNT 2, RenCa, RIN-5F, RMA/RMAS, Saos-2 cells, Sf-9, SkBr3,
T2, T-47D, T84, THP1 cell line, U373, U87, U937, VCaP, Vero cells,
WM39, WT-49, X63, YAC-1, YAR, and transgenic varieties thereof.
Cell lines are available from a variety of sources known to those
with skill in the art (see, e.g., the American Type Culture
Collection (ATCC) (Manassas, Va.)). In some embodiments, a cell
transfected with one or more vectors described herein is used to
establish a new cell line comprising one or more vector-derived
sequences. In some embodiments, a cell transiently transfected with
the components of a CRISPR system as described herein (such as by
transient transfection of one or more vectors, or transfection with
RNA), and modified through the activity of a CRISPR complex, is
used to establish a new cell line comprising cells containing the
modification but lacking any other exogenous sequence. In some
embodiments, cells transiently or non-transiently transfected with
one or more vectors described herein, or cell lines derived from
such cells are used in assessing one or more test compounds.
[0588] In some embodiments, one or more vectors described herein
are used to produce a non-human transgenic animal or transgenic
plant. In some embodiments, the transgenic animal is a mammal, such
as a mouse, rat, or rabbit. In certain embodiments, the organism or
subject is a plant. In certain embodiments, the organism or subject
or plant is algae. Methods for producing transgenic plants and
animals are known in the art, and generally begin with a method of
cell transfection, such as described herein.
[0589] In one aspect, the invention provides for methods of
modifying a target polynucleotide in a eukaryotic cell. In some
embodiments, the method comprises allowing a CRISPR complex to bind
to the target polynucleotide to effect cleavage of said target
polynucleotide thereby modifying the target polynucleotide, wherein
the CRISPR complex comprises a CRISPR enzyme complexed with a guide
sequence hybridized to a target sequence within said target
polynucleotide, wherein said guide sequence is linked to a tracr
mate sequence which in turn hybridizes to a tracr sequence.
[0590] In one aspect, the invention provides a method of modifying
expression of a polynucleotide in a eukaryotic cell. In some
embodiments, the method comprises allowing a CRISPR complex to bind
to the polynucleotide such that said binding results in increased
or decreased expression of said polynucleotide; wherein the CRISPR
complex comprises a CRISPR enzyme complexed with a guide sequence
hybridized to a target sequence within said polynucleotide, wherein
said guide sequence is linked to a tracr mate sequence which in
turn hybridizes to a tracr sequence.
[0591] With recent advances in crop genomics, the ability to use
CRISPR-Cas systems to perform efficient and cost effective gene
editing and manipulation will allow the rapid selection and
comparison of single and multiplexed genetic manipulations to
transform such genomes for improved production and enhanced traits.
In this regard reference is made to US patents and publications:
U.S. Pat. No. 6,603,061--Agrobacterium-Mediated Plant
Transformation Method; U.S. Pat. No. 7,868,149--Plant Genome
Sequences and Uses Thereof and US 2009/0100536--Transgenic Plants
with Enhanced Agronomic Traits, all the contents and disclosure of
each of which are herein incorporated by reference in their
entirety. In the practice of the invention, the contents and
disclosure of Morrell et al "Crop genomics:advances and
applications" Nat Rev Genet. 2011 Dec. 29; 13(2):85-96 are also
herein incorporated by reference in their entirety. In an
advantageous embodiment of the invention, the CRISPR/Cas9 system is
used to engineer microalgae. Accordingly, reference herein to
animal cells may also apply, mutatis mutandis, to plant cells
unless otherwise apparent.
[0592] The practice of the present invention employs, unless
otherwise indicated, conventional techniques of immunology,
biochemistry, chemistry, molecular biology, microbiology, cell
biology, genomics and recombinant DNA, which are within the skill
of the art. See Sambrook, Fritsch and Maniatis, MOLECULAR CLONING:
A LABORATORY MANUAL, 2nd edition (1989); CURRENT PROTOCOLS IN
MOLECULAR BIOLOGY (F. M. Ausubel, et al. eds., (1987)); the series
METHODS IN ENZYMOLOGY (Academic Press, Inc.): PCR 2: A PRACTICAL
APPROACH (M. J. MacPherson, B. D. Hames and G. R. Taylor eds.
(1995)), Harlow and Lane, eds. (1988) ANTIBODIES, A LABORATORY
MANUAL, and ANIMAL CELL CULTURE (R. I. Freshney, ed. (1987)).
Models of Genetic and Epigenetic Conditions
[0593] A method of the invention may be used to create a plant, an
animal or cell that may be used to model and/or study genetic or
epigenetic conditions of interest, such as a through a model of
mutations of interest or a as a disease model. As used herein,
"disease" refers to a disease, disorder, or indication in a
subject. For example, a method of the invention may be used to
create an animal or cell that comprises a modification in one or
more nucleic acid sequences associated with a disease, or a plant,
animal or cell in which the expression of one or more nucleic acid
sequences associated with a disease are altered. Such a nucleic
acid sequence may encode a disease associated protein sequence or
may be a disease associated control sequence. Accordingly, it is
understood that in embodiments of the invention, a plant, subject,
patient, organism or cell can be a non-human subject, patient,
organism or cell. Thus, the invention provides a plant, animal or
cell, produced by the present methods, or a progeny thereof. The
progeny may be a clone of the produced plant or animal, or may
result from sexual reproduction by crossing with other individuals
of the same species to introgress further desirable traits into
their offspring. The cell may be in vivo or ex vivo in the cases of
multicellular organisms, particularly animals or plants. In the
instance where the cell is in cultured, a cell line may be
established if appropriate culturing conditions are met and
preferably if the cell is suitably adapted for this purpose (for
instance a stem cell). Bacterial cell lines produced by the
invention are also envisaged. Hence, cell lines are also
envisaged.
[0594] In some methods, the disease model can be used to study the
effects of mutations on the animal or cell and development and/or
progression of the disease using measures commonly used in the
study of the disease. Alternatively, such a disease model is useful
for studying the effect of a pharmaceutically active compound on
the disease.
[0595] In some methods, the disease model can be used to assess the
efficacy of a potential gene therapy strategy. That is, a
disease-associated gene or polynucleotide can be modified such that
the disease development and/or progression is inhibited or reduced.
In particular, the method comprises modifying a disease-associated
gene or polynucleotide such that an altered protein is produced
and, as a result, the animal or cell has an altered response.
Accordingly, in some methods, a genetically modified animal may be
compared with an animal predisposed to development of the disease
such that the effect of the gene therapy event may be assessed.
[0596] In another embodiment, this invention provides a method of
developing a biologically active agent that modulates a cell
signaling event associated with a disease gene. The method
comprises contacting a test compound with a cell comprising one or
more vectors that drive expression of one or more of a CRISPR
enzyme, a guide sequence linked to a tracr mate sequence, and a
tracr sequence; and detecting a change in a readout that is
indicative of a reduction or an augmentation of a cell signaling
event associated with, e.g., a mutation in a disease gene contained
in the cell.
[0597] A cell model or animal model can be constructed in
combination with the method of the invention for screening a
cellular function change. Such a model may be used to study the
effects of a genome sequence modified by the CRISPR complex of the
invention on a cellular function of interest. For example, a
cellular function model may be used to study the effect of a
modified genome sequence on intracellular signaling or
extracellular signaling. Alternatively, a cellular function model
may be used to study the effects of a modified genome sequence on
sensory perception. In some such models, one or more genome
sequences associated with a signaling biochemical pathway in the
model are modified.
[0598] Several disease models have been specifically investigated.
These include de novo autism risk genes CHD8, KATNAL2, and SCN2A;
and the syndromic autism (Angelman Syndrome) gene UBE3A. These
genes and resulting autism models are of course preferred, but
serve to show the broad applicability of the invention across genes
and corresponding models.
[0599] An altered expression of one or more genome sequences
associated with a signaling biochemical pathway can be determined
by assaying for a difference in the mRNA levels of the
corresponding genes between the test model cell and a control cell,
when they are contacted with a candidate agent. Alternatively, the
differential expression of the sequences associated with a
signaling biochemical pathway is determined by detecting a
difference in the level of the encoded polypeptide or gene
product.
[0600] To assay for an agent-induced alteration in the level of
mRNA transcripts or corresponding polynucleotides, nucleic acid
contained in a sample is first extracted according to standard
methods in the art. For instance, mRNA can be isolated using
various lytic enzymes or chemical solutions according to the
procedures set forth in Sambrook et al. (1989), or extracted by
nucleic-acid-binding resins following the accompanying instructions
provided by the manufacturers. The mRNA contained in the extracted
nucleic acid sample is then detected by amplification procedures or
conventional hybridization assays (e.g. Northern blot analysis)
according to methods widely known in the art or based on the
methods exemplified herein.
[0601] For purpose of this invention, amplification means any
method employing a primer and a polymerase capable of replicating a
target sequence with reasonable fidelity. Amplification may be
carried out by natural or recombinant DNA polymerases such as
TaqGold.TM., T7 DNA polymerase, Klenow fragment of E. coli DNA
polymerase, and reverse transcriptase. A preferred amplification
method is PCR. In particular, the isolated RNA can be subjected to
a reverse transcription assay that is coupled with a quantitative
polymerase chain reaction (RT-PCR) in order to quantify the
expression level of a sequence associated with a signaling
biochemical pathway.
[0602] Detection of the gene expression level can be conducted in
real time in an amplification assay. In one aspect, the amplified
products can be directly visualized with fluorescent DNA-binding
agents including but not limited to DNA intercalators and DNA
groove binders. Because the amount of the intercalators
incorporated into the double-stranded DNA molecules is typically
proportional to the amount of the amplified DNA products, one can
conveniently determine the amount of the amplified products by
quantifying the fluorescence of the intercalated dye using
conventional optical systems in the art. DNA-binding dye suitable
for this application include SYBR green, SYBR blue, DAPI, propidium
iodine, Hoeste, SYBR gold, ethidium bromide, acridines, proflavine,
acridine orange, acriflavine, fluorcoumanin, ellipticine,
daunomycin, chloroquine, distamycin D, chromomycin, homidium,
mithramycin, ruthenium polypyridyls, anthramycin, and the like.
[0603] In another aspect, other fluorescent labels such as sequence
specific probes can be employed in the amplification reaction to
facilitate the detection and quantification of the amplified
products. Probe-based quantitative amplification relies on the
sequence-specific detection of a desired amplified product. It
utilizes fluorescent, target-specific probes (e.g., TaqMan.TM.
probes) resulting in increased specificity and sensitivity. Methods
for performing probe-based quantitative amplification are well
established in the art and are taught in U.S. Pat. No.
5,210,015.
[0604] In yet another aspect, conventional hybridization assays
using hybridization probes that share sequence homology with
sequences associated with a signaling biochemical pathway can be
performed. Typically, probes are allowed to form stable complexes
with the sequences associated with a signaling biochemical pathway
contained within the biological sample derived from the test
subject in a hybridization reaction. It will be appreciated by one
of skill in the art that where antisense is used as the probe
nucleic acid, the target polynucleotides provided in the sample are
chosen to be complementary to sequences of the antisense nucleic
acids. Conversely, where the nucleotide probe is a sense nucleic
acid, the target polynucleotide is selected to be complementary to
sequences of the sense nucleic acid.
[0605] Hybridization can be performed under conditions of various
stringency. Suitable hybridization conditions for the practice of
the present invention are such that the recognition interaction
between the probe and sequences associated with a signaling
biochemical pathway is both sufficiently specific and sufficiently
stable. Conditions that increase the stringency of a hybridization
reaction are widely known and published in the art. See, for
example, (Sambrook, et al., (1989); Nonradioactive In Situ
Hybridization Application Manual, Boehringer Mannheim, second
edition). The hybridization assay can be formed using probes
immobilized on any solid support, including but are not limited to
nitrocellulose, glass, silicon, and a variety of gene arrays. A
preferred hybridization assay is conducted on high-density gene
chips as described in U.S. Pat. No. 5,445,934.
[0606] For a convenient detection of the probe-target complexes
formed during the hybridization assay, the nucleotide probes are
conjugated to a detectable label. Detectable labels suitable for
use in the present invention include any composition detectable by
photochemical, biochemical, spectroscopic, immunochemical,
electrical, optical or chemical means. A wide variety of
appropriate detectable labels are known in the art, which include
fluorescent or chemiluminescent labels, radioactive isotope labels,
enzymatic or other ligands. In preferred embodiments, one will
likely desire to employ a fluorescent label or an enzyme tag, such
as digoxigenin, .beta.-galactosidase, urease, alkaline phosphatase
or peroxidase, avidin/biotin complex.
[0607] The detection methods used to detect or quantify the
hybridization intensity will typically depend upon the label
selected above. For example, radiolabels may be detected using
photographic film or a phosphoimager. Fluorescent markers may be
detected and quantified using a photodetector to detect emitted
light. Enzymatic labels are typically detected by providing the
enzyme with a substrate and measuring the reaction product produced
by the action of the enzyme on the substrate; and finally
colorimetric labels are detected by simply visualizing the colored
label.
[0608] An agent-induced change in expression of sequences
associated with a signaling biochemical pathway can also be
determined by examining the corresponding gene products.
Determining the protein level typically involves a) contacting the
protein contained in a biological sample with an agent that
specifically bind to a protein associated with a signaling
biochemical pathway; and (b) identifying any agent:protein complex
so formed. In one aspect of this embodiment, the agent that
specifically binds a protein associated with a signaling
biochemical pathway is an antibody, preferably a monoclonal
antibody.
[0609] The reaction is performed by contacting the agent with a
sample of the proteins associated with a signaling biochemical
pathway derived from the test samples under conditions that will
allow a complex to form between the agent and the proteins
associated with a signaling biochemical pathway. The formation of
the complex can be detected directly or indirectly according to
standard procedures in the art. In the direct detection method, the
agents are supplied with a detectable label and unreacted agents
may be removed from the complex; the amount of remaining label
thereby indicating the amount of complex formed. For such method,
it is preferable to select labels that remain attached to the
agents even during stringent washing conditions. It is preferable
that the label does not interfere with the binding reaction. In the
alternative, an indirect detection procedure may use an agent that
contains a label introduced either chemically or enzymatically. A
desirable label generally does not interfere with binding or the
stability of the resulting agent:polypeptide complex. However, the
label is typically designed to be accessible to an antibody for an
effective binding and hence generating a detectable signal.
[0610] A wide variety of labels suitable for detecting protein
levels are known in the art. Non-limiting examples include
radioisotopes, enzymes, colloidal metals, fluorescent compounds,
bioluminescent compounds, and chemiluminescent compounds.
[0611] The amount of agent:polypeptide complexes formed during the
binding reaction can be quantified by standard quantitative assays.
As illustrated above, the formation of agent:polypeptide complex
can be measured directly by the amount of label remained at the
site of binding. In an alternative, the protein associated with a
signaling biochemical pathway is tested for its ability to compete
with a labeled analog for binding sites on the specific agent. In
this competitive assay, the amount of label captured is inversely
proportional to the amount of protein sequences associated with a
signaling biochemical pathway present in a test sample.
[0612] A number of techniques for protein analysis based on the
general principles outlined above are available in the art. They
include but are not limited to radioimmunoassays, ELISA (enzyme
linked immunoradiometric assays), "sandwich" immunoassays,
immunoradiometric assays, in situ immunoassays (using e.g.,
colloidal gold, enzyme or radioisotope labels), western blot
analysis, immunoprecipitation assays, immunofluorescent assays, and
SDS-PAGE.
[0613] Antibodies that specifically recognize or bind to proteins
associated with a signaling biochemical pathway are preferable for
conducting the aforementioned protein analyses. Where desired,
antibodies that recognize a specific type of post-translational
modifications (e.g., signaling biochemical pathway inducible
modifications) can be used. Post-translational modifications
include but are not limited to glycosylation, lipidation,
acetylation, and phosphorylation. These antibodies may be purchased
from commercial vendors. For example, anti-phosphotyrosine
antibodies that specifically recognize tyrosine-phosphorylated
proteins are available from a number of vendors including
Invitrogen and Perkin Elmer. Anti-phosphotyrosine antibodies are
particularly useful in detecting proteins that are differentially
phosphorylated on their tyrosine residues in response to an ER
stress. Such proteins include but are not limited to eukaryotic
translation initiation factor 2 alpha (eIF-2a). Alternatively,
these antibodies can be generated using conventional polyclonal or
monoclonal antibody technologies by immunizing a host animal or an
antibody-producing cell with a target protein that exhibits the
desired post-translational modification.
[0614] In practicing the subject method, it may be desirable to
discern the expression pattern of an protein associated with a
signaling biochemical pathway in different bodily tissue, in
different cell types, and/or in different subcellular structures.
These studies can be performed with the use of tissue-specific,
cell-specific or subcellular structure specific antibodies capable
of binding to protein markers that are preferentially expressed in
certain tissues, cell types, or subcellular structures.
[0615] An altered expression of a gene associated with a signaling
biochemical pathway can also be determined by examining a change in
activity of the gene product relative to a control cell. The assay
for an agent-induced change in the activity of a protein associated
with a signaling biochemical pathway will dependent on the
biological activity and/or the signal transduction pathway that is
under investigation. For example, where the protein is a kinase, a
change in its ability to phosphorylate the downstream substrate(s)
can be determined by a variety of assays known in the art.
Representative assays include but are not limited to immunoblotting
and immunoprecipitation with antibodies such as
anti-phosphotyrosine antibodies that recognize phosphorylated
proteins. In addition, kinase activity can be detected by high
throughput chemiluminescent assays such as AlphaScreen.TM.
(available from Perkin Elmer) and eTag.TM. assay (Chan-Hui, et al.
(2003) Clinical Immunology 111: 162-174).
[0616] Where the protein associated with a signaling biochemical
pathway is part of a signaling cascade leading to a fluctuation of
intracellular pH condition, pH sensitive molecules such as
fluorescent pH dyes can be used as the reporter molecules. In
another example where the protein associated with a signaling
biochemical pathway is an ion channel, fluctuations in membrane
potential and/or intracellular ion concentration can be monitored.
A number of commercial kits and high-throughput devices are
particularly suited for a rapid and robust screening for modulators
of ion channels. Representative instruments include FLIPR.TM.
(Molecular Devices, Inc.) and VIPR (Aurora Biosciences). These
instruments are capable of detecting reactions in over 1000 sample
wells of a microplate simultaneously, and providing real-time
measurement and functional data within a second or even a
minisecond.
[0617] In practicing any of the methods disclosed herein, a
suitable vector can be introduced to a cell or an embryo via one or
more methods known in the art, including without limitation,
microinjection, electroporation, sonoporation, biolistics, calcium
phosphate-mediated transfection, cationic transfection, liposome
transfection, dendrimer transfection, heat shock transfection,
nucleofection transfection, magnetofection, lipofection,
impalefection, optical transfection, proprietary agent-enhanced
uptake of nucleic acids, and delivery via liposomes,
immunoliposomes, virosomes, or artificial virions. In some methods,
the vector is introduced into an embryo by microinjection. The
vector or vectors may be microinjected into the nucleus or the
cytoplasm of the embryo. In some methods, the vector or vectors may
be introduced into a cell by nucleofection.
[0618] The target polynucleotide of a CRISPR complex can be any
polynucleotide endogenous or exogenous to the eukaryotic cell. For
example, the target polynucleotide can be a polynucleotide residing
in the nucleus of the eukaryotic cell. The target polynucleotide
can be a sequence coding a gene product (e.g., a protein) or a
non-coding sequence (e.g., a regulatory polynucleotide or a junk
DNA).
[0619] Examples of target polynucleotides include a sequence
associated with a signaling biochemical pathway, e.g., a signaling
biochemical pathway-associated gene or polynucleotide. Examples of
target polynucleotides include a disease associated gene or
polynucleotide. A "disease-associated" gene or polynucleotide
refers to any gene or polynucleotide which is yielding
transcription or translation products at an abnormal level or in an
abnormal form in cells derived from a disease-affected tissues
compared with tissues or cells of a non disease control. It may be
a gene that becomes expressed at an abnormally high level; it may
be a gene that becomes expressed at an abnormally low level, where
the altered expression correlates with the occurrence and/or
progression of the disease. A disease-associated gene also refers
to a gene possessing mutation(s) or genetic variation that is
directly responsible or is in linkage disequilibrium with a gene(s)
that is responsible for the etiology of a disease. The transcribed
or translated products may be known or unknown, and may be at a
normal or abnormal level.
[0620] The target polynucleotide of a CRISPR complex can be any
polynucleotide endogenous or exogenous to the eukaryotic cell. For
example, the target polynucleotide can be a polynucleotide residing
in the nucleus of the eukaryotic cell. The target polynucleotide
can be a sequence coding a gene product (e.g., a protein) or a
non-coding sequence (e.g., a regulatory polynucleotide or a junk
DNA). Without wishing to be bound by theory, it is believed that
the target sequence should be associated with a PAM (protospacer
adjacent motif); that is, a short sequence recognized by the CRISPR
complex. The precise sequence and length requirements for the PAM
differ depending on the CRISPR enzyme used, but PAMs are typically
2-5 base pair sequences adjacent the protospacer (that is, the
target sequence) Examples of PAM sequences are given in the
examples section below, and the skilled person will be able to
identify further PAM sequences for use with a given CRISPR
enzyme.
[0621] The target polynucleotide of a CRISPR complex may include a
number of disease-associated genes and polynucleotides as well as
signaling biochemical pathway-associated genes and polynucleotides
as listed in U.S. provisional patent applications 61/736,527 and
61/748,427 both entitled SYSTEMS METHODS AND COMPOSITIONS FOR
SEQUENCE MANIPULATION filed on Dec. 12, 2012 and Jan. 2, 2013,
respectively, and PCT Application PCT/US2013/074667, entitled
DELIVERY, ENGINEERING AND OPTIMIZATION OF SYSTEMS, METHODS AND
COMPOSITIONS FOR SEQUENCE MANIPULATION AND THERAPEUTIC
APPLICATIONS, filed Dec. 12, 2013, the contents of all of which are
herein incorporated by reference in their entirety.
[0622] Examples of target polynucleotides include a sequence
associated with a signaling biochemical pathway, e.g., a signaling
biochemical pathway-associated gene or polynucleotide. Examples of
target polynucleotides include a disease associated gene or
polynucleotide. A "disease-associated" gene or polynucleotide
refers to any gene or polynucleotide which is yielding
transcription or translation products at an abnormal level or in an
abnormal form in cells derived from a disease-affected tissues
compared with tissues or cells of a non disease control. It may be
a gene that becomes expressed at an abnormally high level; it may
be a gene that becomes expressed at an abnormally low level, where
the altered expression correlates with the occurrence and/or
progression of the disease. A disease-associated gene also refers
to a gene possessing mutation(s) or genetic variation that is
directly responsible or is in linkage disequilibrium with a gene(s)
that is responsible for the etiology of a disease. The transcribed
or translated products may be known or unknown, and may be at a
normal or abnormal level.
Genome-Wide Knock-Out Screening
[0623] The CRISPR-Cas9 proteins and systems described herein can be
used to perform efficient and cost effective functional genomic
screens. Such screens can utilize CRISPR-Cas9 genome wide
libraries. Such screens and libraries can provide for determining
the function of genes, cellular pathways genes are involved in, and
how any alteration in gene expression can result in a particular
biological process. An advantage of the present invention is that
the CRISPR system avoids off-target binding and its resulting side
effects. This is achieved using systems arranged to have a high
degree of sequence specificity for the target DNA.
[0624] A genome wide library may comprise a plurality of CRISPR-Cas
system guide RNAs, as described herein, comprising guide sequences
that are capable of targeting a plurality of target sequences in a
plurality of genomic loci in a population of eukaryotic cells. The
population of cells may be a population of embryonic stem (ES)
cells. The target sequence in the genomic locus may be a non-coding
sequence. The non-coding sequence may be an intron, regulatory
sequence, splice site, 3' UTR, 5' UTR, or polyadenylation signal.
Gene function of one or more gene products may be altered by said
targeting. The targeting may result in a knockout of gene function.
The targeting of a gene product may comprise more than one guide
RNA. A gene product may be targeted by 2, 3, 4, 5, 6, 7, 8, 9, or
10 guide RNAs, preferably 3 to 4 per gene. Off-target modifications
may be minimized by exploiting the staggered double strand breaks
generated by Cas9 effector protein complexes or by utilizing
methods analogous to those used in CRISPR-Cas9 systems. (See, e.g.,
DNA targeting specificity of RNA-guided Cas9 nucleases. Hsu, P.,
Scott, D., Weinstein, J., Ran, F A., Konermann, S., Agarwala, V.,
Li, Y., Fine, E., Wu, X., Shalem, O., Cradick, T J., Marraffini, L
A., Bao, G., & Zhang, F. Nat Biotechnol doi:10.1038/nbt.2647
(2013)), incorporated herein by reference. The targeting may be of
about 100 or more sequences. The targeting may be of about 1000 or
more sequences. The targeting may be of about 20,000 or more
sequences. The targeting may be of the entire genome. The targeting
may be of a panel of target sequences focused on a relevant or
desirable pathway. The pathway may be an immune pathway. The
pathway may be a cell division pathway.
[0625] One aspect of the invention comprehends a genome wide
library that may comprise a plurality of CRISPR-Cas system guide
RNAs that may comprise guide sequences that are capable of
targeting a plurality of target sequences in a plurality of genomic
loci, wherein said targeting results in a knockout of gene
function. This library may potentially comprise guide RNAs that
target each and every gene in the genome of an organism.
[0626] In some embodiments of the invention the organism or subject
is a eukaryote (including mammal including human) or a non-human
eukaryote or a non-human animal or a non-human mammal. In some
embodiments, the organism or subject is a non-human animal, and may
be an arthropod, for example, an insect, or may be a nematode. In
some methods of the invention the organism or subject is a plant.
In some methods of the invention the organism or subject is a
mammal or a non-human mammal. A non-human mammal may be for example
a rodent (preferably a mouse or a rat), an ungulate, or a primate.
In some methods of the invention the organism or subject is algae,
including microalgae, or is a fungus.
[0627] The knockout of gene function may comprise: introducing into
each cell in the population of cells a vector system of one or more
vectors comprising an engineered, non-naturally occurring
CRISPR-Cas system comprising I. a Cas9 protein, and II. one or more
guide RNAs, wherein components I and II may be same or on different
vectors of the system, integrating components I and II into each
cell, wherein the guide sequence targets a unique gene in each
cell, wherein the Cas9 protein is operably linked to a regulatory
element, wherein when transcribed, the guide RNA comprising the
guide sequence directs sequence-specific binding of a CRISPR-Cas
system to a target sequence in the genomic loci of the unique gene,
inducing cleavage of the genomic loci by the Cas9 protein, and
confirming different knockout mutations in a plurality of unique
genes in each cell of the population of cells thereby generating a
gene knockout cell library. The invention comprehends that the
population of cells is a population of eukaryotic cells, and in a
preferred embodiment, the population of cells is a population of
embryonic stem (ES) cells.
[0628] The one or more vectors may be plasmid vectors. The vector
may be a single vector comprising Cas9, a sgRNA, and optionally, a
selection marker into target cells. Not being bound by a theory,
the ability to simultaneously deliver Cas9 and sgRNA through a
single vector enables application to any cell type of interest,
without the need to first generate cell lines that express Cas9.
The regulatory element may be an inducible promoter. The inducible
promoter may be a doxycycline inducible promoter. In some methods
of the invention the expression of the guide sequence is under the
control of the T7 promoter and is driven by the expression of T7
polymerase. The confirming of different knockout mutations may be
by whole exome sequencing. The knockout mutation may be achieved in
100 or more unique genes. The knockout mutation may be achieved in
1000 or more unique genes. The knockout mutation may be achieved in
20,000 or more unique genes. The knockout mutation may be achieved
in the entire genome. The knockout of gene function may be achieved
in a plurality of unique genes which function in a particular
physiological pathway or condition. The pathway or condition may be
an immune pathway or condition. The pathway or condition may be a
cell division pathway or condition.
[0629] The invention also provides kits that comprise the genome
wide libraries mentioned herein. The kit may comprise a single
container comprising vectors or plasmids comprising the library of
the invention. The kit may also comprise a panel comprising a
selection of unique CRISPR-Cas system guide RNAs comprising guide
sequences from the library of the invention, wherein the selection
is indicative of a particular physiological condition. The
invention comprehends that the targeting is of about 100 or more
sequences, about 1000 or more sequences or about 20,000 or more
sequences or the entire genome. Furthermore, a panel of target
sequences may be focused on a relevant or desirable pathway, such
as an immune pathway or cell division.
[0630] In an additional aspect of the invention, a Cas9 enzyme may
comprise one or more mutations and may be used as a generic DNA
binding protein with or without fusion to a functional domain. The
mutations may be artificially introduced mutations or gain- or
loss-of-function mutations. The mutations may include but are not
limited to mutations in one of the catalytic domains (D10 and H840)
in the RuvC and HNH catalytic domains, respectively. Further
mutations have been characterized. In one aspect of the invention,
the functional domain may be a transcriptional activation domain,
which may be VP64. In other aspects of the invention, the
functional domain may be a transcriptional repressor domain, which
may be KRAB or SID4X. Other aspects of the invention relate to the
mutated Cas 9 enzyme being fused to domains which include but are
not limited to a transcriptional activator, repressor, a
recombinase, a transposase, a histone remodeler, a demethylase, a
DNA methyltransferase, a cryptochrome, a light
inducible/controllable domain or a chemically
inducible/controllable domain. Some methods of the invention can
include inducing expression of targeted genes. In one embodiment,
inducing expression by targeting a plurality of target sequences in
a plurality of genomic loci in a population of eukaryotic cells is
by use of a functional domain.
[0631] Useful in the practice of the instant invention, reference
is made to: [0632] Genome-Scale CRISPR-Cas9 Knockout Screening in
Human Cells. Shalem, O., Sanjana, N E., Hartenian, E., Shi, X.,
Scott, D A., Mikkelson, T., Heckl, D., Ebert, B L., Root, D E.,
Doench, J G., Zhang, F. Science December 12. (2013). [Epub ahead of
print]; Published in final edited form as: Science. 2014 Jan. 3;
343(6166): 84-87. [0633] Shalem et al. involves a new way to
interrogate gene function on a genome-wide scale. Their studies
showed that delivery of a genome-scale CRISPR-Cas9 knockout (GeCKO)
library targeted 18,080 genes with 64,751 unique guide sequences
enabled both negative and positive selection screening in human
cells. First, the authors showed use of the GeCKO library to
identify genes essential for cell viability in cancer and
pluripotent stem cells. Next, in a melanoma model, the authors
screened for genes whose loss is involved in resistance to
vemurafenib, a therapeutic that inhibits mutant protein kinase
BRAF. Their studies showed that the highest-ranking candidates
included previously validated genes NF1 and MED12 as well as novel
hits NF2, CUL3, TADA2B, and TADA1. The authors observed a high
level of consistency between independent guide RNAs targeting the
same gene and a high rate of hit confirmation, and thus
demonstrated the promise of genome-scale screening with Cas9.
[0634] Reference is also made to US patent publication number
US20140357530; and PCT Patent Publication WO2014093701, hereby
incorporated herein by reference. Reference is also made to NIH
Press Release of Oct. 22, 2015 entitled, "Researchers identify
potential alternative to CRISPR-Cas genome editing tools: New Cas
enzymes shed light on evolution of CRISPR-Cas systems, which is
incorporated by reference.
Functional Alteration and Screening
[0635] In another aspect, the present invention provides for a
method of functional evaluation and screening of genes. The use of
the CRISPR system of the present invention to precisely deliver
functional domains, to activate or repress genes or to alter
epigenetic state by precisely altering the methylation site on a
specific locus of interest, can be with one or more guide RNAs
applied to a single cell or population of cells or with a library
applied to genome in a pool of cells ex vivo or in vivo comprising
the administration or expression of a library comprising a
plurality of guide RNAs (sgRNAs) and wherein the screening further
comprises use of a Cas9 effector protein, wherein the CRISPR
complex comprising the Cas9 effector protein is modified to
comprise a heterologous functional domain. In an aspect the
invention provides a method for screening a genome comprising the
administration to a host or expression in a host in vivo of a
library. In an aspect the invention provides a method as herein
discussed further comprising an activator administered to the host
or expressed in the host. In an aspect the invention provides a
method as herein discussed wherein the activator is attached to a
Cas9 effector protein. In an aspect the invention provides a method
as herein discussed wherein the activator is attached to the N
terminus or the C terminus of the Cas9 effector protein. In an
aspect the invention provides a method as herein discussed wherein
the activator is attached to a sgRNA loop. In an aspect the
invention provides a method as herein discussed further comprising
a repressor administered to the host or expressed in the host. In
an aspect the invention provides a method as herein discussed,
wherein the screening comprises affecting and detecting gene
activation, gene inhibition, or cleavage in the locus.
[0636] In an aspect, the invention provides efficient on-target
activity and minimizes off target activity. In an aspect, the
invention provides efficient on-target cleavage by Cas9 effector
protein and minimizes off-target cleavage by the Cas9 effector
protein. In an aspect, the invention provides guide specific
binding of Cas9 effector protein at a gene locus without DNA
cleavage. Accordingly, in an aspect, the invention provides
target-specific gene regulation. In an aspect, the invention
provides guide specific binding of Cas9 effector protein at a gene
locus without DNA cleavage. Accordingly, in an aspect, the
invention provides for cleavage at one gene locus and gene
regulation at a different gene locus using a single Cas9 effector
protein. In an aspect, the invention provides orthogonal activation
and/or inhibition and/or cleavage of multiple targets using one or
more Cas9 effector protein and/or enzyme.
[0637] In an aspect the invention provides a method as herein
discussed, wherein the host is a eukaryotic cell. In an aspect the
invention provides a method as herein discussed, wherein the host
is a mammalian cell. In an aspect the invention provides a method
as herein discussed, wherein the host is a non-human eukaryote. In
an aspect the invention provides a method as herein discussed,
wherein the non-human eukaryote is a non-human mammal. In an aspect
the invention provides a method as herein discussed, wherein the
non-human mammal is a mouse. An aspect the invention provides a
method as herein discussed comprising the delivery of the Cas9
effector protein complexes or component(s) thereof or nucleic acid
molecule(s) coding therefor, wherein said nucleic acid molecule(s)
are operatively linked to regulatory sequence(s) and expressed in
vivo. In an aspect the invention provides a method as herein
discussed wherein the expressing in vivo is via a lentivirus, an
adenovirus, or an AAV. In an aspect the invention provides a method
as herein discussed wherein the delivery is via a particle, a
nanoparticle, a lipid or a cell penetrating peptide (CPP).
[0638] In an aspect the invention provides a pair of CRISPR
complexes comprising Cas9 effector protein, each comprising a guide
RNA (sgRNA) comprising a guide sequence capable of hybridizing to a
target sequence in a genomic locus of interest in a cell, wherein
at least one loop of each sgRNA is modified by the insertion of
distinct RNA sequence(s) that bind to one or more adaptor proteins,
and wherein the adaptor protein is associated with one or more
functional domains, wherein each sgRNA of each Cas9 effector
protein complex comprises a functional domain having a DNA cleavage
activity. In an aspect the invention provides paired Cas9 effector
protein complexes as herein-discussed, wherein the DNA cleavage
activity is due to a Fok1 nuclease.
[0639] In an aspect the invention provides a method for cutting a
target sequence in a genomic locus of interest comprising delivery
to a cell of the Cas9 effector protein complexes or component(s)
thereof or nucleic acid molecule(s) coding therefor, wherein said
nucleic acid molecule(s) are operatively linked to regulatory
sequence(s) and expressed in vivo. In an aspect the invention
provides a method as herein-discussed wherein the delivery is via a
lentivirus, an adenovirus, or an AAV. In an aspect the invention
provides a method as herein-discussed or paired Cas9 effector
protein complexes as herein-discussed wherein the target sequence
for a first complex of the pair is on a first strand of double
stranded DNA and the target sequence for a second complex of the
pair is on a second strand of double stranded DNA. In an aspect the
invention provides a method as herein-discussed or paired Cas9
effector protein complexes as herein-discussed wherein the target
sequences of the first and second complexes are in proximity to
each other such that the DNA is cut in a manner that facilitates
homology directed repair. In an aspect a herein method can further
include introducing into the cell template DNA. In an aspect a
herein method or herein paired Cas9 effector protein complexes can
involve wherein each Cas9 effector protein complex has a Cas9
effector enzyme that is mutated such that it has no more than about
5% of the nuclease activity of the Cas9 effector enzyme that is not
mutated.
[0640] In an aspect the invention provides a library, method or
complex as herein-discussed wherein the sgRNA is modified to have
at least one non-coding functional loop, e.g., wherein the at least
one non-coding functional loop is repressive; for instance, wherein
the at least one non-coding functional loop comprises Alu.
[0641] In one aspect, the invention provides a method for altering
or modifying expression of a gene product. The said method may
comprise introducing into a cell containing and expressing a DNA
molecule encoding the gene product an engineered, non-naturally
occurring CRISPR system comprising a Cas9 effector protein and
guide RNA that targets the DNA molecule, whereby the guide RNA
targets the DNA molecule encoding the gene product and the Cas9
effector protein cleaves the DNA molecule encoding the gene
product, whereby expression of the gene product is altered; and,
wherein the Cas9 effector protein and the guide RNA do not
naturally occur together. The invention comprehends the guide RNA
comprising a guide sequence linked to a direct repeat sequence. The
invention further comprehends the Cas9 effector protein being codon
optimized for expression in a Eukaryotic cell. In a preferred
embodiment the Eukaryotic cell is a mammalian cell and in a more
preferred embodiment the mammalian cell is a human cell. In a
further embodiment of the invention, the expression of the gene
product is decreased.
[0642] In some embodiments, one or more functional domains are
associated with the CRISPR enzyme, for example a Type II Cas9
enzyme.
[0643] In some embodiments, one or more functional domains are
associated with an adaptor protein, for example as used with the
modified guides of Konnerman et al. (Nature 517, 583-588, 29 Jan.
2015).
[0644] In some embodiments, one or more functional domains are
associated with an dead sgRNA (dRNA). In some embodiments, a dRNA
complex with active cas9 directs gene regulation by a functional
domain at on gene locus while an sgRNA directs DNA cleavage by the
active cas9 at another locus, for example as described by Dahlman
et al., `Orthogonal gene control with a catalytically active Cas9
nuclease` (in press). In some embodiments, dRNAs are selected to
maximize selectivity of regulation for a gene locus of interest
compared to off-target regulation. In some embodiments, dRNAs are
selected to maximize target gene regulation and minimize target
cleavage
[0645] For the purposes of the following discussion, reference to a
functional domain could be a functional domain associated with the
CRISPR enzyme or a functional domain associated with the adaptor
protein.
[0646] In the practice of the invention, loops of the sgRNA may be
extended, without colliding with the Cas9 protein by the insertion
of distinct RNA loop(s) or distinct sequence(s) that may recruit
adaptor proteins that can bind to the distinct RNA loop(s) or
distinct sequence(s). The adaptor proteins may include but are not
limited to orthogonal RNA-binding protein/aptamer combinations that
exist within the diversity of bacteriophage coat proteins. A list
of such coat proteins includes, but is not limited to: Q.beta., F2,
GA, fr, JP501, M12, R17, BZ13, JP34, JP500, KU1, M11, MX1, TW18,
VK, SP, FI, ID2, NL95, TW19, AP205, .phi.Cb5, .phi.Cb8r,
.phi.Cb12r, .phi.Cb23r, 7s and PRR1. These adaptor proteins or
orthogonal RNA binding proteins can further recruit effector
proteins or fusions which comprise one or more functional domains.
In some embodiments, the functional domain may be selected from the
group consisting of: transposase domain, integrase domain,
recombinase domain, resolvase domain, invertase domain, protease
domain, DNA methyltransferase domain, DNA hydroxylmethylase domain,
DNA demethylase domain, histone acetylase domain, histone
deacetylases domain, nuclease domain, repressor domain, activator
domain, nuclear-localization signal domains,
transcription-regulatory protein (or transcription complex
recruiting) domain, cellular uptake activity associated domain,
nucleic acid binding domain, antibody presentation domain, histone
modifying enzymes, recruiter of histone modifying enzymes;
inhibitor of histone modifying enzymes, histone methyltransferase,
histone demethylase, histone kinase, histone phosphatase, histone
ribosylase, histone deribosylase, histone ubiquitinase, histone
deubiquitinase, histone biotinase and histone tail protease. In
some preferred embodiments, the functional domain is a
transcriptional activation domain, such as, without limitation,
VP64, p65, MyoD1, HSF1, RTA, SET7/9 or a histone acetyltransferase.
In some embodiments, the functional domain is a transcription
repression domain, preferably KRAB. In some embodiments, the
transcription repression domain is SID, or concatemers of SID (eg
SID4X). In some embodiments, the functional domain is an epigenetic
modifying domain, such that an epigenetic modifying enzyme is
provided. In some embodiments, the functional domain is an
activation domain, which may be the P65 activation domain.
Saturating Mutagenesis
[0647] CRISPR-Cas9 System(s) can be used to perform saturating or
deep scanning mutagenesis of genomic loci in conjunction with a
cellular phenotype--for instance, for determining critical minimal
features and discrete vulnerabilities of functional elements
required for gene expression, drug resistance, and reversal of
disease. By saturating or deep scanning mutagenesis is meant that
every or essentially every DNA base is cut within the genomic loci.
A library of CRISPR-Cas guide RNAs may be introduced into a
population of cells. The library may be introduced, such that each
cell receives a single guide RNA (sgRNA). In the case where the
library is introduced by transduction of a viral vector, as
described herein, a low multiplicity of infection (MOI) is used.
The library may include sgRNAs targeting every sequence upstream of
a (protospacer adjacent motif) (PAM) sequence in a genomic locus.
The library may include at least 100 non-overlapping genomic
sequences upstream of a PAM sequence for every 1000 base pairs
within the genomic locus. The library may include sgRNAs targeting
sequences upstream of at least one different PAM sequence. The
CRISPR-Cas System(s) may include more than one Cas protein. Any Cas
protein as described herein, including orthologues or engineered
Cas proteins that recognize different PAM sequences may be used.
The frequency of off target sites for a sgRNA may be less than 500.
Off target scores may be generated to select sgRNAs with the lowest
off target sites. Any phenotype determined to be associated with
cutting at a sgRNA target site may be confirmed by using sgRNA's
targeting the same site in a single experiment. Validation of a
target site may also be performed by using a nickase Cas9, as
described herein, and two sgRNAs targeting the genomic site of
interest. Not being bound by a theory, a target site is a true hit
if the change in phenotype is observed in validation
experiments.
[0648] The genomic loci may include at least one continuous genomic
region. The at least one continuous genomic region may comprise up
to the entire genome. The at least one continuous genomic region
may comprise a functional element of the genome. The functional
element may be within a non-coding region, coding gene, intronic
region, promoter, or enhancer. The at least one continuous genomic
region may comprise at least 1 kb, preferably at least 50 kb of
genomic DNA. The at least one continuous genomic region may
comprise a transcription factor binding site. The at least one
continuous genomic region may comprise a region of DNase I
hypersensitivity. The at least one continuous genomic region may
comprise a transcription enhancer or repressor element. The at
least one continuous genomic region may comprise a site enriched
for an epigenetic signature. The at least one continuous genomic
DNA region may comprise an epigenetic insulator. The at least one
continuous genomic region may comprise two or more continuous
genomic regions that physically interact. Genomic regions that
interact may be determined by `4C technology`. 4C technology allows
the screening of the entire genome in an unbiased manner for DNA
segments that physically interact with a DNA fragment of choice, as
is described in Zhao et al. ((2006) Nat Genet 38, 1341-7) and in
U.S. Pat. No. 8,642,295, both incorporated herein by reference in
its entirety. The epigenetic signature may be histone acetylation,
histone methylation, histone ubiquitination, histone
phosphorylation, DNA methylation, or a lack thereof
[0649] CRISPR-Cas System(s) for saturating or deep scanning
mutagenesis can be used in a population of cells. The CRISPR-Cas
System(s) can be used in eukaryotic cells, including but not
limited to mammalian and plant cells. The population of cells may
be prokaryotic cells. The population of eukaryotic cells may be a
population of embryonic stem (ES) cells, neuronal cells, epithelial
cells, immune cells, endocrine cells, muscle cells, erythrocytes,
lymphocytes, plant cells, or yeast cells.
[0650] In one aspect, the present invention provides for a method
of screening for functional elements associated with a change in a
phenotype. The library may be introduced into a population of cells
that are adapted to contain a Cas protein. The cells may be sorted
into at least two groups based on the phenotype. The phenotype may
be expression of a gene, cell growth, or cell viability. The
relative representation of the guide RNAs present in each group are
determined, whereby genomic sites associated with the change in
phenotype are determined by the representation of guide RNAs
present in each group. The change in phenotype may be a change in
expression of a gene of interest. The gene of interest may be
upregulated, downregulated, or knocked out. The cells may be sorted
into a high expression group and a low expression group. The
population of cells may include a reporter construct that is used
to determine the phenotype. The reporter construct may include a
detectable marker. Cells may be sorted by use of the detectable
marker.
[0651] In another aspect, the present invention provides for a
method of screening for genomic sites associated with resistance to
a chemical compound. The chemical compound may be a drug or
pesticide. The library may be introduced into a population of cells
that are adapted to contain a Cas protein, wherein each cell of the
population contains no more than one guide RNA; the population of
cells are treated with the chemical compound; and the
representation of guide RNAs are determined after treatment with
the chemical compound at a later time point as compared to an early
time point, whereby genomic sites associated with resistance to the
chemical compound are determined by enrichment of guide RNAs.
Representation of sgRNAs may be determined by deep sequencing
methods.
[0652] Useful in the practice of the instant invention, reference
is made to the article entitled BCL11A enhancer dissection by
Cas9-mediated in situ saturating mutagenesis. Canver, M. C., Smith,
E. C., Sher, F., Pinello, L., Sanjana, N. E., Shalem, O., Chen, D.
D., Schupp, P. G., Vinjamur, D. S., Garcia, S. P., Luc, S., Kurita,
R., Nakamura, Y., Fujiwara, Y., Maeda, T., Yuan, G., Zhang, F.,
Orkin, S. H., & Bauer, D. E. DOI:10.1038/nature15521, published
online Sep. 16, 2015, the article is herein incorporated by
reference and discussed briefly below: [0653] Canver et al.
describes novel pooled CRISPR-Cas9 guide RNA libraries to perform
in situ saturating mutagenesis of the human and mouse BCL11A
erythroid enhancers previously identified as an enhancer associated
with fetal hemoglobin (HbF) level and whose mouse ortholog is
necessary for erythroid BCL11A expression. This approach revealed
critical minimal features and discrete vulnerabilities of these
enhancers. Through editing of primary human progenitors and mouse
transgenesis, the authors validated the BCL11A erythroid enhancer
as a target for HbF reinduction. The authors generated a detailed
enhancer map that informs therapeutic genome editing.
Method of Using CRISPR-Cas9 Systems to Modify a Cell or
Organism
[0654] The invention in some embodiments comprehends a method of
modifying a cell or organism. The cell may be a prokaryotic cell or
a eukaryotic cell. The cell may be a mammalian cell. The mammalian
cell many be a non-human primate, bovine, porcine, rodent or mouse
cell. The cell may be a non-mammalian eukaryotic cell such as
poultry, fish or shrimp. The cell may also be a plant cell. The
plant cell may be of a crop plant such as cassava, corn, sorghum,
wheat, or rice. The plant cell may also be of an algae, tree or
vegetable. The modification introduced to the cell by the present
invention may be such that the cell and progeny of the cell are
altered for improved production of biologic products such as an
antibody, starch, alcohol or other desired cellular output. The
modification introduced to the cell by the present invention may be
such that the cell and progeny of the cell include an alteration
that changes the biologic product produced.
[0655] The system may comprise one or more different vectors. In an
aspect of the invention, the Cas protein is codon optimized for
expression the desired cell type, preferentially a eukaryotic cell,
preferably a mammalian cell or a human cell.
[0656] Packaging cells are typically used to form virus particles
that are capable of infecting a host cell. Such cells include 293
cells, which package adenovirus, and .psi.2 cells or PA317 cells,
which package retrovirus. Viral vectors used in gene therapy are
usually generated by producing a cell line that packages a nucleic
acid vector into a viral particle. The vectors typically contain
the minimal viral sequences required for packaging and subsequent
integration into a host, other viral sequences being replaced by an
expression cassette for the polynucleotide(s) to be expressed. The
missing viral functions are typically supplied in trans by the
packaging cell line. For example, AAV vectors used in gene therapy
typically only possess ITR sequences from the AAV genome which are
required for packaging and integration into the host genome. Viral
DNA is packaged in a cell line, which contains a helper plasmid
encoding the other AAV genes, namely rep and cap, but lacking ITR
sequences. The cell line may also be infected with adenovirus as a
helper. The helper virus promotes replication of the AAV vector and
expression of AAV genes from the helper plasmid. The helper plasmid
is not packaged in significant amounts due to a lack of ITR
sequences. Contamination with adenovirus can be reduced by, e.g.,
heat treatment to which adenovirus is more sensitive than AAV.
Additional methods for the delivery of nucleic acids to cells are
known to those skilled in the art. See, for example, US20030087817,
incorporated herein by reference.
[0657] In some embodiments, a host cell is transiently or
non-transiently transfected with one or more vectors described
herein. In some embodiments, a cell is transfected as it naturally
occurs in a subject. In some embodiments, a cell that is
transfected is taken from a subject. In some embodiments, the cell
is derived from cells taken from a subject, such as a cell line. A
wide variety of cell lines for tissue culture are known in the art.
Examples of cell lines include, but are not limited to, C8161,
CCRF-CEM, MOLT, mIMCD-3, NHDF, HeLa-S3, Huh1, Huh4, Huh7, HUVEC,
HASMC, HEKn, HEKa, MiaPaCell, Panc1, PC-3, TF1, CTLL-2, C1R, Rat6,
CV1, RPTE, A10, T24, J82, A375, ARH-77, Calu1, SW480, SW620, SKOV3,
SK-UT, CaCo2, P388D1, SEM-K2, WEHI-231, HB56, TIB55, Jurkat,
J45.01, LRMB, Bcl-1, BC-3, IC21, DLD2, Raw264.7, NRK, NRK-52E,
MRC5, MEF, Hep G2, HeLa B, HeLa T4, COS, COS-1, COS-6, COS-M6A,
BS-C-1 monkey kidney epithelial, BALB/3T3 mouse embryo fibroblast,
3T3 Swiss, 3T3-L1, 132-d5 human fetal fibroblasts; 10.1 mouse
fibroblasts, 293-T, 3T3, 721, 9L, A2780, A2780ADR, A2780cis, A172,
A20, A253, A431, A-549, ALC, B16, B35, BCP-1 cells, BEAS-2B,
bEnd.3, BHK-21, BR 293, BxPC3, C3H-10T1/2, C6/36, Cal-27, CHO,
CHO-7, CHO-IR, CHO-K1, CHO-K2, CHO-T, CHO Dhfr -/-, COR-L23,
COR-L23/CPR, COR-L23/5010, COR-L23/R23, COS-7, COV-434, CML T1,
CMT, CT26, D17, DH82, DU145, DuCaP, EL4, EM2, EM3, EMT6/AR1,
EMT6/AR10.0, FM3, H1299, H69, HB54, HB55, HCA2, HEK-293, HeLa,
Hepa1c1c7, HL-60, HMEC, HT-29, Jurkat, JY cells, K562 cells, Ku812,
KCL22, KG1, KYO1, LNCap, Ma-Mel 1-48, MC-38, MCF-7, MCF-10A,
MDA-MB-231, MDA-MB-468, MDA-MB-435, MDCK II, MDCK II, MOR/0.2R,
MONO-MAC 6, MTD-1A, MyEnd, NCI-H69/CPR, NCI-H69/LX10, NCI-H69/LX20,
NCI-H69/LX4, NIH-3T3, NALM-1, NW-145, OPCN/OPCT cell lines, Peer,
PNT-1A/PNT 2, RenCa, RIN-5F, RMA/RMAS, Saos-2 cells, Sf-9, SkBr3,
T2, T-47D, T84, THP1 cell line, U373, U87, U937, VCaP, Vero cells,
WM39, WT-49, X63, YAC-1, YAR, and transgenic varieties thereof.
Cell lines are available from a variety of sources known to those
with skill in the art (see, e.g., the American Type Culture
Collection (ATCC) (Manassas, Va.)). In some embodiments, a cell
transfected with one or more vectors described herein is used to
establish a new cell line comprising one or more vector-derived
sequences. In some embodiments, a cell transiently transfected with
the components of a nucleic acid-targeting system as described
herein (such as by transient transfection of one or more vectors,
or transfection with RNA), and modified through the activity of a
nucleic acid-targeting complex, is used to establish a new cell
line comprising cells containing the modification but lacking any
other exogenous sequence. In some embodiments, cells transiently or
non-transiently transfected with one or more vectors described
herein, or cell lines derived from such cells are used in assessing
one or more test compounds.
[0658] In some embodiments, one or more vectors described herein
are used to produce a non-human transgenic animal or transgenic
plant. In some embodiments, the transgenic animal is a mammal, such
as a mouse, rat, or rabbit. In certain embodiments, the organism or
subject is a plant. In certain embodiments, the organism or subject
or plant is algae. Methods for producing transgenic plants and
animals are known in the art, and generally begin with a method of
cell transfection, such as described herein.
[0659] In one aspect, the invention provides for methods of
modifying a target polynucleotide in a eukaryotic cell. In some
embodiments, the method comprises allowing a nucleic acid-targeting
complex to bind to the target polynucleotide to effect cleavage of
said target polynucleotide thereby modifying the target
polynucleotide, wherein the nucleic acid-targeting complex
comprises a nucleic acid-targeting effector protein complexed with
a guide RNA hybridized to a target sequence within said target
polynucleotide.
[0660] In one aspect, the invention provides a method of modifying
expression of a polynucleotide in a eukaryotic cell. In some
embodiments, the method comprises allowing a nucleic acid-targeting
complex to bind to the polynucleotide such that said binding
results in increased or decreased expression of said
polynucleotide; wherein the nucleic acid-targeting complex
comprises a nucleic acid-targeting effector protein complexed with
a guide RNA hybridized to a target sequence within said
polynucleotide.
CRISPR Systems can be Used in Plants
[0661] CRISPR-Cas system(s) (e.g., single or multiplexed) can be
used in conjunction with recent advances in crop genomics. Such
CRISPR-Cas system(s) can be used to perform efficient and cost
effective plant gene or genome interrogation or editing or
manipulation--for instance, for rapid investigation and/or
selection and/or interrogations and/or comparison and/or
manipulations and/or transformation of plant genes or genomes;
e.g., to create, identify, develop, optimize, or confer trait(s) or
characteristic(s) to plant(s) or to transform a plant genome. There
can accordingly be improved production of plants, new plants with
new combinations of traits or characteristics or new plants with
enhanced traits. Such CRISPR-Cas system(s) can be used with regard
to plants in Site-Directed Integration (SDI) or Gene Editing (GE)
or any Near Reverse Breeding (NRB) or Reverse Breeding (RB)
techniques. With respect to use of the CRISPR-Cas system in plants,
mention is made of the University of Arizona website "CRISPR-PLANT"
(http://www.genome.arizona.edu/crispr/) (supported by Penn State
and AGI). Embodiments of the invention can be used in genome
editing in plants or where RNAi or similar genome editing
techniques have been used previously; see, e.g., Nekrasov, "Plant
genome editing made easy: targeted mutagenesis in model and crop
plants using the CRISPR/Cas system," Plant Methods 2013, 9:39
(doi:10.1186/1746-4811-9-39); Brooks, "Efficient gene editing in
tomato in the first generation using the CRISPR/Cas9 system," Plant
Physiology September 2014 pp 114.247577; Shan, "Targeted genome
modification of crop plants using a CRISPR-Cas system," Nature
Biotechnology 31, 686-688 (2013); Feng, "Efficient genome editing
in plants using a CRISPR/Cas system," Cell Research (2013)
23:1229-1232. doi:10.1038/cr.2013.114; published online 20 Aug.
2013; Xie, "RNA-guided genome editing in plants using a CRISPR-Cas
system," Mol Plant. 2013 November; 6(6):1975-83. doi:
10.1093/mp/sst119. Epub 2013 Aug. 17; Xu, "Gene targeting using the
Agrobacterium tumefaciens-mediated CRISPR-Cas system in rice," Rice
2014, 7:5 (2014), Zhou et al., "Exploiting SNPs for biallelic
CRISPR mutations in the outcrossing woody perennial Populus reveals
4-coumarate: CoA ligase specificity and Redundancy," New
Phytologist (2015) (Forum) 1-4 (available online only at
www.newphytologist.com); Caliando et al, "Targeted DNA degradation
using a CRISPR device stably carried in the host genome, NATURE
COMMUNICATIONS 6:6989, DOI: 10.1038/ncomms7989,
www.nature.com/naturecommunications DOI: 10.1038/ncomms7989; U.S.
Pat. No. 6,603,061--Agrobacterium-Mediated Plant Transformation
Method; U.S. Pat. No. 7,868,149--Plant Genome Sequences and Uses
Thereof and US 2009/0100536--Transgenic Plants with Enhanced
Agronomic Traits, all the contents and disclosure of each of which
are herein incorporated by reference in their entirety. In the
practice of the invention, the contents and disclosure of Morrell
et al "Crop genomics: advances and applications," Nat Rev Genet.
2011 Dec. 29; 13(2):85-96; each of which is incorporated by
reference herein including as to how herein embodiments may be used
as to plants. Accordingly, reference herein to animal cells may
also apply, mutatis mutandis, to plant cells unless otherwise
apparent; and, the enzymes herein having reduced off-target effects
and systems employing such enzymes can be used in plant
applications, including those mentioned herein.
[0662] Sugano et al. (Plant Cell Physiol. 2014 March; 55(3):475-81.
doi: 10.1093/pcp/pcu014. Epub 2014 Jan. 18) reports the application
of CRISPR/Cas9 to targeted mutagenesis in the liverwort Marchantia
polymorpha L., which has emerged as a model species for studying
land plant evolution. The U6 promoter of M. polymorpha was
identified and cloned to express the gRNA. The target sequence of
the gRNA was designed to disrupt the gene encoding auxin response
factor 1 (ARF1) in M. polymorpha. Using Agrobacterium-mediated
transformation, Sugano et al. isolated stable mutants in the
gametophyte generation of M. polymorpha. CRISPR/Cas9-based
site-directed mutagenesis in vivo was achieved using either the
Cauliflower mosaic virus 35S or M. polymorpha EF1.alpha. promoter
to express Cas9. Isolated mutant individuals showing an
auxin-resistant phenotype were not chimeric. Moreover, stable
mutants were produced by asexual reproduction of T1 plants.
Multiple arf1 alleles were easily established using
CRIPSR/Cas9-based targeted mutagenesis. The methods of Sugano et
al. may be applied to the CRISPR Cas system of the present
invention.
[0663] Kabadi et al. (Nucleic Acids Res. 2014 Oct. 29; 42(19):e147.
doi: 10.1093/nar/gku749. Epub 2014 Aug. 13) developed a single
lentiviral system to express a Cas9 variant, a reporter gene and up
to four sgRNAs from independent RNA polymerase III promoters that
are incorporated into the vector by a convenient Golden Gate
cloning method. Each sgRNA was efficiently expressed and can
mediate multiplex gene editing and sustained transcriptional
activation in immortalized and primary human cells. The methods of
Kabadi et al. may be applied to the CRISPR Cas system of the
present invention.
[0664] Ling et al. (BMC Plant Biology 2014, 14:327) developed a
CRISPR/Cas9 binary vector set based on the pGreen or pCAMBIA
backbone, as well as a gRNA This toolkit requires no restriction
enzymes besides BsaI to generate final constructs harboring
maize-codon optimized Cas9 and one or more gRNAs with high
efficiency in as little as one cloning step. The toolkit was
validated using maize protoplasts, transgenic maize lines, and
transgenic Arabidopsis lines and was shown to exhibit high
efficiency and specificity. More importantly, using this toolkit,
targeted mutations of three Arabidopsis genes were detected in
transgenic seedlings of the T1 generation. Moreover, the
multiple-gene mutations could be inherited by the next generation.
(guide RNA) module vector set, as a toolkit for multiplex genome
editing in plants. The toolbox of Lin et al. may be applied to the
CRISPR Cas system of the present invention.
[0665] Protocols for targeted plant genome editing via CRISPR/Cas9
are also available in volume 1284 of the series Methods in
Molecular Biology pp 239-255 10 Feb. 2015. A detailed procedure to
design, construct, and evaluate dual gRNAs for plant codon
optimized Cas9 (pcoCas9) mediated genome editing using Arabidopsis
thaliana and Nicotiana benthamiana protoplasts s model cellular
systems are described. Strategies to apply the CRISPR/Cas9 system
to generating targeted genome modifications in whole plants are
also discussed. The protocols described in the chapter may be
applied to the CRISPR Cas system of the present invention.
[0666] Ma et al. (Mol Plant. 2015 Aug. 3; 8(8):1274-84. doi:
10.1016/j.molp.2015.04.007) reports robust CRISPR/Cas9 vector
system, utilizing a plant codon optimized Cas9 gene, for convenient
and high-efficiency multiplex genome editing in monocot and dicot
plants. Ma et al. designed PCR-based procedures to rapidly generate
multiple sgRNA expression cassettes, which can be assembled into
the binary CRISPR/Cas9 vectors in one round of cloning by Golden
Gate ligation or Gibson Assembly. With this system, Ma et al.
edited 46 target sites in rice with an average 85.4% rate of
mutation, mostly in biallelic and homozygous status. Ma et al.
provide examples of loss-of-function gene mutations in T0 rice and
T1Arabidopsis plants by simultaneous targeting of multiple (up to
eight) members of a gene family, multiple genes in a biosynthetic
pathway, or multiple sites in a single gene. The methods of Ma et
al. may be applied to the CRISPR Cas system of the present
invention.
[0667] Lowder et al. (Plant Physiol. 2015 Aug. 21. pii: pp.
00636.2015) also developed a CRISPR/Cas9 toolbox enables multiplex
genome editing and transcriptional regulation of expressed,
silenced or non-coding genes in plants. This toolbox provides
researchers with a protocol and reagents to quickly and efficiently
assemble functional CRISPR/Cas9 T-DNA constructs for monocots and
dicots using Golden Gate and Gateway cloning methods. It comes with
a full suite of capabilities, including multiplexed gene editing
and transcriptional activation or repression of plant endogenous
genes. T-DNA based transformation technology is fundamental to
modern plant biotechnology, genetics, molecular biology and
physiology. As such, Applicants developed a method for the assembly
of Cas9 (WT, nickase or dCas9) and gRNA(s) into a T-DNA
destination-vector of interest. The assembly method is based on
both Golden Gate assembly and MultiSite Gateway recombination.
Three modules are required for assembly. The first module is a Cas9
entry vector, which contains promoterless Cas9 or its derivative
genes flanked by attL1 and attR5 sites. The second module is a gRNA
entry vector which contains entry gRNA expression cassettes flanked
by attL5 and attL2 sites. The third module includes
attR1-attR2-containing destination T-DNA vectors that provide
promoters of choice for Cas9 expression. The toolbox of Lowder et
al. may be applied to the CRISPR Cas system of the present
invention.
[0668] In an advantageous embodiment, the plant may be a tree. The
present invention may also utilize the herein disclosed CRISPR Cas
system for herbaceous systems (see, e.g., Belhaj et al., Plant
Methods 9: 39 and Harrison et al., Genes & Development 28:
1859-1872). In a particularly advantageous embodiment, the CRISPR
Cas system of the present invention may target single nucleotide
polymorphisms (SNPs) in trees (see, e.g., Zhou et al., New
Phytologist, Volume 208, Issue 2, pages 298-301, October 2015). In
the Zhou et al. study, the authors applied a CRISPR Cas system in
the woody perennial Populus using the 4-coumarate:CoA ligase (4CL)
gene family as a case study and achieved 100% mutational efficiency
for two 4CL genes targeted, with every transformant examined
carrying biallelic modifications. In the Zhou et al., study, the
CRISPR/Cas9 system was highly sensitive to single nucleotide
polymorphisms (SNPs), as cleavage for a third 4CL gene was
abolished due to SNPs in the target sequence.
[0669] The methods of Zhou et al. (New Phytologist, Volume 208,
Issue 2, pages 298-301, October 2015) may be applied to the present
invention as follows. Two 4CL genes, 4CL1 and 4CL2, associated with
lignin and flavonoid biosynthesis, respectively are targeted for
CRISPR/Cas9 editing. The Populus tremula.times.alba clone 717-1B4
routinely used for transformation is divergent from the
genome-sequenced Populus trichocarpa. Therefore, the 4CL1 and 4CL2
gRNAs designed from the reference genome are interrogated with
in-house 717 RNA-Seq data to ensure the absence of SNPs which could
limit Cas efficiency. A third gRNA designed for 4CL5, a genome
duplicate of 4CL1, is also included. The corresponding 717 sequence
harbors one SNP in each allele near/within the PAM, both of which
are expected to abolish targeting by the 4CL5-gRNA. All three gRNA
target sites are located within the first exon. For 717
transformation, the gRNA is expressed from the Medicago U6.6
promoter, along with a human codon-optimized Cas under control of
the CaMV 35S promoter in a binary vector. Transformation with the
Cas-only vector can serve as a control. Randomly selected 4CL1 and
4CL2 lines are subjected to amplicon-sequencing. The data is then
processed and biallelic mutations are confirmed in all cases.
[0670] In plants, pathogens are often host-specific. For example,
Fusarium oxysporum f. sp. lycopersici causes tomato wilt but
attacks only tomato, and F. oxysporum f. dianthii Puccinia graminis
f sp. tritici attacks only wheat. Plants have existing and induced
defenses to resist most pathogens. Mutations and recombination
events across plant generations lead to genetic variability that
gives rise to susceptibility, especially as pathogens reproduce
with more frequency than plants. In plants there can be non-host
resistance, e.g., the host and pathogen are incompatible. There can
also be Horizontal Resistance, e.g., partial resistance against all
races of a pathogen, typically controlled by many genes and
Vertical Resistance, e.g., complete resistance to some races of a
pathogen but not to other races, typically controlled by a few
genes. In a Gene-for-Gene level, plants and pathogens evolve
together, and the genetic changes in one balance changes in other.
Accordingly, using Natural Variability, breeders combine most
useful genes for Yield, Quality, Uniformity, Hardiness, Resistance.
The sources of resistance genes include native or foreign
Varieties, Heirloom Varieties, Wild Plant Relatives, and Induced
Mutations, e.g., treating plant material with mutagenic agents.
Using the present invention, plant breeders are provided with a new
tool to induce mutations. Accordingly, one skilled in the art can
analyze the genome of sources of resistance genes, and in Varieties
having desired characteristics or traits employ the present
invention to induce the rise of resistance genes, with more
precision than previous mutagenic agents and hence accelerate and
improve plant breeding programs.
CRISPR Systems can be Used in Non-Human Organisms/Animals
[0671] The present application may also be extended to other
agricultural applications such as, for example, farm and production
animals. For example, pigs have many features that make them
attractive as biomedical models, especially in regenerative
medicine. In particular, pigs with severe combined immunodeficiency
(SCID) may provide useful models for regenerative medicine,
xenotransplantation, and tumor development and will aid in
developing therapies for human SCID patients. Lee et al., (Proc
Natl Acad Sci USA. 2014 May 20; 111(20):7260-5) utilized a
reporter-guided transcription activator-like effector nuclease
(TALEN) system to generated targeted modifications of recombination
activating gene (RAG) 2 in somatic cells at high efficiency,
including some that affected both alleles. CRISPR Cas may be
applied to a similar system.
[0672] The methods of Lee et al., (Proc Natl Acad Sci USA. 2014 May
20; 111(20):7260-5) may be applied to the present invention as
follows. Mutated pigs are produced by targeted modification of RAG2
in fetal fibroblast cells followed by SCNT and embryo transfer.
Constructs coding for CRISPR Cas and a reporter are electroporated
into fetal-derived fibroblast cells. After 48 h, transfected cells
expressing the green fluorescent protein are sorted into individual
wells of a 96-well plate at an estimated dilution of a single cell
per well. Targeted modification of RAG2 are screened by amplifying
a genomic DNA fragment flanking any CRISPR Cas cutting sites
followed by sequencing the PCR products. After screening and
ensuring lack of off-site mutations, cells carrying targeted
modification of RAG2 are used for SCNT. The polar body, along with
a portion of the adjacent cytoplasm of oocyte, presumably
containing the metaphase II plate, are removed, and a donor cell
are placed in the perivitelline. The reconstructed embryos are then
electrically porated to fuse the donor cell with the oocyte and
then chemically activated. The activated embryos are incubated in
Porcine Zygote Medium 3 (PZM3) with 0.5 .mu.M Scriptaid (S7817;
Sigma-Aldrich) for 14-16 h. Embryos are then washed to remove the
Scriptaid and cultured in PZM3 until they were transferred into the
oviducts of surrogate pigs.
[0673] The present invention is also applicable to modifying SNPs
of other animals, such as cows. Tan et al. (Proc Natl Acad Sci USA.
2013 Oct. 8; 110(41): 16526-16531) expanded the livestock gene
editing toolbox to include transcription activator-like (TAL)
effector nuclease (TALEN)- and clustered regularly interspaced
short palindromic repeats (CRISPR)/Cas9-stimulated
homology-directed repair (HDR) using plasmid, rAAV, and
oligonucleotide templates. Gene specific gRNA sequences were cloned
into the Church lab gRNA vector (Addgene ID: 41824) according to
their methods (Mali P, et al. (2013) RNA-Guided Human Genome
Engineering via Cas9. Science 339(6121):823-826). The Cas9 nuclease
was provided either by co-transfection of the hCas9 plasmid
(Addgene ID: 41815) or mRNA synthesized from RCIScript-hCas9. This
RCIScript-hCas9 was constructed by sub-cloning the XbaI-AgeI
fragment from the hCas9 plasmid (encompassing the hCas9 cDNA) into
the RCIScript plasmid.
[0674] Heo et al. (Stem Cells Dev. 2015 Feb. 1; 24(3):393-402. doi:
10.1089/scd.2014.0278. Epub 2014 Nov. 3) reported highly efficient
gene targeting in the bovine genome using bovine pluripotent cells
and clustered regularly interspaced short palindromic repeat
(CRISPR)/Cas9 nuclease. First, Heo et al. generate induced
pluripotent stem cells (iPSCs) from bovine somatic fibroblasts by
the ectopic expression of yamanaka factors and GSK3.beta. and MEK
inhibitor (2i) treatment. Heo et al. observed that these bovine
iPSCs are highly similar to naive pluripotent stem cells with
regard to gene expression and developmental potential in teratomas.
Moreover, CRISPR/Cas9 nuclease, which was specific for the bovine
NANOG locus, showed highly efficient editing of the bovine genome
in bovine iPSCs and embryos.
[0675] Igenity.RTM. provides a profile analysis of animals, such as
cows, to perform and transmit traits of economic traits of economic
importance, such as carcass composition, carcass quality, maternal
and reproductive traits and average daily gain. The analysis of a
comprehensive Igenity.RTM. profile begins with the discovery of DNA
markers (most often single nucleotide polymorphisms or SNPs). All
the markers behind the Igenity.RTM. profile were discovered by
independent scientists at research institutions, including
universities, research organizations, and government entities such
as USDA. Markers are then analyzed at Igenity.RTM. in validation
populations. Igenity.RTM. uses multiple resource populations that
represent various production environments and biological types,
often working with industry partners from the seedstock, cow-calf,
feedlot and/or packing segments of the beef industry to collect
phenotypes that are not commonly available. Cattle genome databases
are widely available, see, e.g., the NAGRP Cattle Genome
Coordination Program
(http://www.animalgenome.org/cattle/maps/db.html). Thus, the
present invention maybe applied to target bovine SNPs. One of skill
in the art may utilize the above protocols for targeting SNPs and
apply them to bovine SNPs as described, for example, by Tan et al.
or Heo et al.
Therapeutic Targeting with RNA-Guided Effector Protein Complex
[0676] As will be apparent, it is envisaged that the present system
can be used to target any polynucleotide sequence of interest. The
invention provides a non-naturally occurring or engineered
composition, or one or more polynucleotides encoding components of
said composition, or vector or delivery systems comprising one or
more polynucleotides encoding components of said composition for
use in a modifying a target cell in vivo, ex vivo or in vitro and,
may be conducted in a manner alters the cell such that once
modified the progeny or cell line of the CRISPR modified cell
retains the altered phenotype. The modified cells and progeny may
be part of a multi-cellular organism such as a plant or animal with
ex vivo or in vivo application of CRISPR system to desired cell
types. The CRISPR invention may be a therapeutic method of
treatment. The therapeutic method of treatment may comprise gene or
genome editing, or gene therapy.
Treating Pathogens, Like Bacterial, Fungal and Parasitic
Pathogens
[0677] The present invention may also be applied to treat
bacterial, fungal and parasitic pathogens. Most research efforts
have focused on developing new antibiotics, which once developed,
would nevertheless be subject to the same problems of drug
resistance. The invention provides novel CRISPR-based alternatives
which overcome those difficulties. Furthermore, unlike existing
antibiotics, CRISPR-based treatments can be made pathogen specific,
inducing bacterial cell death of a target pathogen while avoiding
beneficial bacteria.
[0678] Jiang et al. ("RNA-guided editing of bacterial genomes using
CRISPR-Cas systems," Nature Biotechnology vol. 31, p. 233-9, March
2013) used a CRISPR-Cas9 system to mutate or kill S. pneumoniae and
E. coli. The work, which introduced precise mutations into the
genomes, relied on dual-RNA:Cas9-directed cleavage at the targeted
genomic site to kill unmutated cells and circumvented the need for
selectable markers or counter-selection systems. CRISPR systems
have be used to reverse antibiotic resistance and eliminate the
transfer of resistance between strains. Bickard et al. showed that
Cas9, reprogrammed to target virulence genes, kills virulent, but
not avirulent, S. aureus. Reprogramming the nuclease to target
antibiotic resistance genes destroyed staphylococcal plasmids that
harbor antibiotic resistance genes and immunized against the spread
of plasmid-borne resistance genes. (see, Bikard et al., "Exploiting
CRISPR-Cas nucleases to produce sequence-specific antimicrobials,"
Nature Biotechnology vol. 32, 1146-1150, doi:10.1038/nbt.3043,
published online 5 Oct. 2014.) Bikard showed that CRISPR-Cas9
antimicrobials function in vivo to kill S. aureus in a mouse skin
colonization model. Similarly, Yosef et al used a CRISPR system to
target genes encoding enzymes that confer resistance to
.beta.-lactam antibiotics (see Yousef et al., "Temperate and lytic
bacteriophages programmed to sensitize and kill
antibiotic-resistant bacteria," Proc. Natl. Acad. Sci. USA, vol.
112, p. 7267-7272, doi: 10.1073/pnas.1500107112 published online
May 18, 2015).
[0679] CRISPR systems can be used to edit genomes of parasites that
are resistant to other genetic approaches. For example, a
CRISPR-Cas9 system was shown to introduce double-stranded breaks
into the in the Plasmodium yoelii genome (see, Zhang et al.,
"Efficient Editing of Malaria Parasite Genome Using the CRISPR/Cas9
System," mBio. vol. 5, e01414-14, July-August 2014). Ghorbal et al.
("Genome editing in the human malaria parasite Plasmodium
falciparumusing the CRISPR-Cas9 system," Nature Biotechnology, vol.
32, p. 819-821, doi: 10.1038/nbt.2925, published online Jun. 1,
2014) modified the sequences of two genes, orc1 and kelchl3, which
have putative roles in gene silencing and emerging resistance to
artemisinin, respectively. Parasites that were altered at the
appropriate sites were recovered with very high efficiency, despite
there being no direct selection for the modification, indicating
that neutral or even deleterious mutations can be generated using
this system. CRISPR-Cas9 is also used to modify the genomes of
other pathogenic parasites, including Toxoplasma gondii (see Shen
et al., "Efficient gene disruption in diverse strains of Toxoplasma
gondii using CRISPR/CAS9," mBio vol. 5:e01114-14, 2014; and Sidik
et al., "Efficient Genome Engineering of Toxoplasma gondii Using
CRISPR/Cas9," PLoS One vol. 9, e100450, doi:
10.1371/journal.pone.0100450, published online Jun. 27, 2014).
[0680] Vyas et al. ("A Candida albicans CRISPR system permits
genetic engineering of essential genes and gene families," Science
Advances, vol. 1, e1500248, DOI: 10.1126/sciadv.1500248, Apr. 3,
2015) employed a CRISPR system to overcome long-standing obstacles
to genetic engineering in C. albicans and efficiently mutate in a
single experiment both copies of several different genes. In an
organism where several mechanisms contribute to drug resistance,
Vyas produced homozygous double mutants that no longer displayed
the hyper-resistance to fluconazole or cycloheximide displayed by
the parental clinical isolate Can90. Vyas also obtained homozygous
loss-of-function mutations in essential genes of C. albicans by
creating conditional alleles. Null alleles of DCR1, which is
required for ribosomal RNA processing, are lethal at low
temperature but viable at high temperature. Vyas used a repair
template that introduced a nonsense mutation and isolated dcr1/dcr1
mutants that failed to grow at 16.degree. C.
[0681] The CRISPR system of the present invention for use in P.
falciparum by disrupting chromosomal loci. Ghorbal et al. ("Genome
editing in the human malaria parasite Plasmodium falciparum using
the CRISPR-Cas9 system", Nature Biotechnology, 32, 819-821 (2014),
DOI: 10.1038/nbt.2925, Jun. 1, 2014) employed a CRISPR system to
introduce specific gene knockouts and single-nucleotide
substitutions in the malaria genome. To adapt the CRISPR-Cas9
system to P. falciparum, Ghorbal et al. generated expression
vectors for under the control of plasmoidal regulatory elements in
the pUF1-Cas9 episome that also carries the drug-selectable marker
ydhodh, which gives resistance to DSM1, a P. falciparum
dihydroorotate dehydrogenase (PfDHODH) inhibitor and for
transcription of the sgRNA, used P. falciparum U6 small nuclear
(sn)RNA regulatory elements placing the guide RNA and the donor DNA
template for homologous recombination repair on the same plasmid,
pL7. See also, Zhang C. et al. ("Efficient editing of malaria
parasite genome using the CRISPR/Cas9 system", MBio, 2014 Jul. 1;
5(4):E01414-14, doi: 10.1128/MbIO.01414-14) and Wagner et al.
("Efficient CRISPR-Cas9-mediated genome editing in Plasmodium
falciparum, Nature Methods 11, 915-918 (2014), DOI:
10.1038/nmeth.3063).
Treating Pathogens, Like Viral Pathogens Such as HIV
[0682] Cas-mediated genome editing might be used to introduce
protective mutations in somatic tissues to combat nongenetic or
complex diseases. For example, NHEJ-mediated inactivation of the
CCR5 receptor in lymphocytes (Lombardo et al., Nat Biotechnol. 2007
November; 25(11):1298-306) may be a viable strategy for
circumventing HIV infection, whereas deletion of PCSK9 (Cohen et
al., Nat Genet. 2005 February; 37(2):161-5) orangiopoietin
(Musunuru et al., N Engl J Med. 2010 Dec. 2; 363(23):2220-7) may
provide therapeutic effects against statin-resistant
hypercholesterolemia or hyperlipidemia. Although these targets may
be also addressed using siRNA-mediated protein knockdown, a unique
advantage of NHEJ-mediated gene inactivation is the ability to
achieve permanent therapeutic benefit without the need for
continuing treatment. As with all gene therapies, it will of course
be important to establish that each proposed therapeutic use has a
favorable benefit-risk ratio.
[0683] Hydrodynamic delivery of plasmid DNA encoding Cas9 and guide
RNA along with a repair template into the liver of an adult mouse
model of tyrosinemia was shown to be able to correct the mutant Fah
gene and rescue expression of the wild-type Fah protein in .about.1
out of 250 cells (Nat Biotechnol. 2014 June; 32(6):551-3). In
addition, clinical trials successfully used ZF nucleases to combat
HIV infection by ex vivo knockout of the CCR5 receptor. In all
patients, HIV DNA levels decreased, and in one out of four
patients, HIV RNA became undetectable (Tebas et al., N Engl J Med.
2014 Mar. 6; 370(10):901-10). Both of these results demonstrate the
promise of programmable nucleases as a new therapeutic
platform.
[0684] In another embodiment, self-inactivating lentiviral vectors
with an siRNA targeting a common exon shared by HIV tat/rev, a
nucleolar-localizing TAR decoy, and an anti-CCR5-specific
hammerhead ribozyme (see, e.g., DiGiusto et al. (2010) Sci Transl
Med 2:36ra43) may be used/and or adapted to the CRISPR-Cas system
of the present invention. A minimum of 2.5.times.10.sup.6 CD34+
cells per kilogram patient weight may be collected and
prestimulated for 16 to 20 hours in X-VIVO 15 medium (Lonza)
containing 2 .mu.mol/L-glutamine, stem cell factor (100 ng/ml),
Flt-3 ligand (Flt-3L) (100 ng/ml), and thrombopoietin (10 ng/ml)
(CellGenix) at a density of 2.times.10.sup.6 cells/ml.
Prestimulated cells may be transduced with lentiviral at a
multiplicity of infection of 5 for 16 to 24 hours in 75-cm.sup.2
tissue culture flasks coated with fibronectin (25 mg/cm.sup.2)
(RetroNectin, Takara Bio Inc.).
[0685] With the knowledge in the art and the teachings in this
disclosure the skilled person can correct HSCs as to
immunodeficiency condition such as HIV/AIDS comprising contacting
an HSC with a CRISPR-Cas9 system that targets and knocks out CCR5.
An guide RNA (and advantageously a dual guide approach, e.g., a
pair of different guide RNAs; for instance, guide RNAs targeting of
two clinically relevant genes, B2M and CCR5, in primary human CD4+
T cells and CD34+ hematopoietic stem and progenitor cells (HSPCs))
that targets and knocks out CCR5-and-Cas9 protein containing
particle is contacted with HSCs. The so contacted cells can be
administered; and optionally treated/expanded; cf. Cartier. See
also Kiem, "Hematopoietic stem cell-based gene therapy for HIV
disease," Cell Stem Cell. Feb. 3, 2012; 10(2): 137-147;
incorporated herein by reference along with the documents it cites;
Mandal et al, "Efficient Ablation of Genes in Human Hematopoietic
Stem and Effector Cells using CRISPR/Cas9," Cell Stem Cell, Volume
15, Issue 5, p643-652, 6 Nov. 2014; incorporated herein by
reference along with the documents it cites. Mention is also made
of Ebina, "CRISPR/Cas9 system to suppress HIV-1 expression by
editing HIV-1 integrated proviral DNA" SCIENTIFIC REPORTS |3:
2510|DOI: 10.1038/srep02510, incorporated herein by reference along
with the documents it cites, as another means for combatting
HIV/AIDS using a CRISPR-Cas9 system.
[0686] The rationale for genome editing for HIV treatment
originates from the observation that individuals homozygous for
loss of function mutations in CCR5, a cellular co-receptor for the
virus, are highly resistant to infection and otherwise healthy,
suggesting that mimicking this mutation with genome editing could
be a safe and effective therapeutic strategy [Liu, R., et al. Cell
86, 367-377 (1996)]. This idea was clinically validated when an HIV
infected patient was given an allogeneic bone marrow transplant
from a donor homozygous for a loss of function CCR5 mutation,
resulting in undetectable levels of HIV and restoration of normal
CD4 T-cell counts [Hutter, G., et al. The New England journal of
medicine 360, 692-698 (2009)]. Although bone marrow transplantation
is not a realistic treatment strategy for most HIV patients, due to
cost and potential graft vs. host disease, HIV therapies that
convert a patient's own T-cells into CCR5 are desirable.
[0687] Early studies using ZFNs and NHEJ to knockout CCR5 in
humanized mouse models of HIV showed that transplantation of CCR5
edited CD4 T cells improved viral load and CD4 T-cell counts
[Perez, E. E., et al. Nature biotechnology 26, 808-816 (2008)].
Importantly, these models also showed that HIV infection resulted
in selection for CCR5 null cells, suggesting that editing confers a
fitness advantage and potentially allowing a small number of edited
cells to create a therapeutic effect.
[0688] As a result of this and other promising preclinical studies,
genome editing therapy that knocks out CCR5 in patient T cells has
now been tested in humans [Holt, N., et al. Nature biotechnology
28, 839-847 (2010); Li, L., et al. Molecular therapy: the journal
of the American Society of Gene Therapy 21, 1259-1269 (2013)]. In a
recent phase I clinical trial, CD4+ T cells from patients with HIV
were removed, edited with ZFNs designed to knockout the CCR5 gene,
and autologously transplanted back into patients [Tebas, P., et al.
The New England journal of medicine 370, 901-910 (2014)].
[0689] In another study (Mandal et al., Cell Stem Cell, Volume 15,
Issue 5, p643-652, 6 Nov. 2014), CRISPR-Cas9 has targeted two
clinical relevant genes, B2M and CCR5, in human CD4+ T cells and
CD34+ hematopoietic stem and progenitor cells (HSPCs). Use of
single RNA guides led to highly efficient mutagenesis in HSPCs but
not in T cells. A dual guide approach improved gene deletion
efficacy in both cell types. HSPCs that had undergone genome
editing with CRISPR-Cas9 retained multilineage potential. Predicted
on- and off-target mutations were examined via target capture
sequencing in HSPCs and low levels of off-target mutagenesis were
observed at only one site. These results demonstrate that
CRISPR-Cas9 can efficiently ablate genes in HSPCs with minimal
off-target mutagenesis, which have broad applicability for
hematopoietic cell-based therapy.
[0690] Wang et al. (PLoS One. 2014 Dec. 26; 9(12):e115987. doi:
10.1371/journal.pone.0115987) silenced CCR5 via CRISPR associated
protein 9 (Cas9) and single guided RNAs (guide RNAs) with
lentiviral vectors expressing Cas9 and CCR5 guide RNAs. Wang et al.
showed that a single round transduction of lentiviral vectors
expressing Cas9 and CCR5 guide RNAs into HIV-1 susceptible human
CD4+ cells yields high frequencies of CCR5 gene disruption. CCR5
gene-disrupted cells are not only resistant to R5-tropic HIV-1,
including transmitted/founder (T/F) HIV-1 isolates, but also have
selective advantage over CCR5 gene-undisrupted cells during
R5-tropic HIV-1 infection. Genome mutations at potential off-target
sites that are highly homologous to these CCR5 guide RNAs in stably
transduced cells even at 84 days post transduction were not
detected by a T7 endonuclease I assay.
[0691] Fine et al. (Sci Rep. 2015 Jul. 1; 5:10777. doi:
10.1038/srep10777) identified a two-cassette system expressing
pieces of the S. pyogenes Cas9 (SpCas9) protein which splice
together in cellula to form a functional protein capable of
site-specific DNA cleavage. With specific CRISPR guide strands,
Fine et al. demonstrated the efficacy of this system in cleaving
the HBB and CCR5 genes in human HEK-293T cells as a single Cas9 and
as a pair of Cas9 nickases. The trans-spliced SpCas9 (tsSpCas9)
displayed .about.35% of the nuclease activity compared with the
wild-type SpCas9 (wtSpCas9) at standard transfection doses, but had
substantially decreased activity at lower dosing levels. The
greatly reduced open reading frame length of the tsSpCas9 relative
to wtSpCas9 potentially allows for more complex and longer genetic
elements to be packaged into an AAV vector including
tissue-specific promoters, multiplexed guide RNA expression, and
effector domain fusions to SpCas9.
[0692] Li et al. (J Gen Virol. 2015 August; 96(8):2381-93. doi:
10.1099/vir.0.000139. Epub 2015 Apr. 8) demonstrated that
CRISPR-Cas9 can efficiently mediate the editing of the CCR5 locus
in cell lines, resulting in the knockout of CCR5 expression on the
cell surface. Next-generation sequencing revealed that various
mutations were introduced around the predicted cleavage site of
CCR5. For each of the three most effective guide RNAs that were
analyzed, no significant off-target effects were detected at the 15
top-scoring potential sites. By constructing chimeric Ad5F35
adenoviruses carrying CRISPR-Cas9 components, Li et al. efficiently
transduced primary CD4+ T-lymphocytes and disrupted CCR5
expression, and the positively transduced cells were conferred with
HIV-1 resistance.
[0693] Mention is made of WO 2015/148670 and through the teachings
herein the invention comprehends methods and materials of this
document applied in conjunction with the teachings herein. In an
aspect of gene therapy, methods and compositions for editing of a
target sequence related to or in connection with Human
Immunodeficiency Virus (HIV) and Acquired Immunodeficiency Syndrome
(AIDS) are comprehended. In a related aspect, the invention
described herein comprehends prevention and treatment of HIV
infection and AIDS, by introducing one or more mutations in the
gene for C-C chemokine receptor type 5 (CCR5). The CCR5 gene is
also known as CKR5, CCR-5, CD195, CKR-5, CCCKR5, CMKBR5, IDDM22,
and CC-CKR-5. In a further aspect, the invention described herein
comprehends provide for prevention or reduction of HIV infection
and/or prevention or reduction of the ability for HIV to enter host
cells, e.g., in subjects who are already infected. Exemplary host
cells for HIV include, but are not limited to, CD4 cells, T cells,
gut associated lymphatic tissue (GALT), macrophages, dendritic
cells, myeloid precursor cell, and microglia. Viral entry into the
host cells requires interaction of the viral glycoproteins gp41 and
gp120 with both the CD4 receptor and a co-receptor, e.g., CCR5. If
a co-receptor, e.g., CCR5, is not present on the surface of the
host cells, the virus cannot bind and enter the host cells. The
progress of the disease is thus impeded. By knocking out or
knocking down CCR5 in the host cells, e.g., by introducing a
protective mutation (such as a CCR5 delta 32 mutation), entry of
the HIV virus into the host cells is prevented.
[0694] One of skill in the art may utilize the above studies of,
for example, Holt, N., et al. Nature biotechnology 28, 839-847
(2010), Li, L., et al. Molecular therapy: the journal of the
American Society of Gene Therapy 21, 1259-1269 (2013), Mandal et
al., Cell Stem Cell, Volume 15, Issue 5, p643-652, 6 Nov. 2014,
Wang et al. (PLoS One. 2014 Dec. 26; 9(12):e115987. doi:
10.1371/journal.pone.0115987), Fine et al. (Sci Rep. 2015 Jul. 1;
5:10777. doi: 10.1038/srep10777) and Li et al. (J Gen Virol. 2015
August; 96(8):2381-93. doi: 10.1099/vir.0.000139. Epub 2015 Apr. 8)
for targeting CCR5 with the CRISPR Cas system of the present
invention.
Treating Pathogens, Like Viral Pathogens, Such as HBV
[0695] The present invention may also be applied to treat hepatitis
B virus (HBV). However, the CRISPR Cas system must be adapted to
avoid the shortcomings of RNAi, such as the risk of oversatring
endogenous small RNA pathways, by for example, optimizing dose and
sequence (see, e.g., Grimm et al., Nature vol. 441, 26 May 2006).
For example, low doses, such as about 1-10.times.10.sup.14
particles per human are contemplated. In another embodiment, the
CRISPR Cas system directed against HBV may be administered in
liposomes, such as a stable nucleic-acid-lipid particle (SNALP)
(see, e.g., Morrissey et al., Nature Biotechnology, Vol. 23, No. 8,
August 2005). Daily intravenous injections of about 1, 3 or 5
mg/kg/day of CRISPR Cas targeted to HBV RNA in a SNALP are
contemplated. The daily treatment may be over about three days and
then weekly for about five weeks. In another embodiment, the system
of Chen et al. (Gene Therapy (2007) 14, 11-19) may be used/and or
adapted for the CRISPR Cas system of the present invention. Chen et
al. use a double-stranded adenoassociated virus 8-pseudotyped
vector (dsAAV2/8) to deliver shRNA. A single administration of
dsAAV2/8 vector (1.times.10.sup.12 vector genomes per mouse),
carrying HBV-specific shRNA, effectively suppressed the steady
level of HBV protein, mRNA and replicative DNA in liver of HBV
transgenic mice, leading to up to 2-3 log.sub.10 decrease in HBV
load in the circulation. Significant HBV suppression sustained for
at least 120 days after vector administration. The therapeutic
effect of shRNA was target sequence dependent and did not involve
activation of interferon. For the present invention, a CRISPR Cas
system directed to HBV may be cloned into an AAV vector, such as a
dsAAV2/8 vector and administered to a human, for example, at a
dosage of about 1.times.10.sup.15 vector genomes to about
1.times.10.sup.16 vector genomes per human. In another embodiment,
the method of Wooddell et al. (Molecular Therapy vol. 21 no. 5,
973-985 May 2013) may be used/and or adapted to the CRISPR Cas
system of the present invention. Woodell et al. show that simple
coinjection of a hepatocyte-targeted,
N-acetylgalactosamine-conjugated melittin-like peptide (NAG-MLP)
with a liver-tropic cholesterol-conjugated siRNA (chol-siRNA)
targeting coagulation factor VII (F7) results in efficient F7
knockdown in mice and nonhuman primates without changes in clinical
chemistry or induction of cytokines. Using transient and transgenic
mouse models of HBV infection, Wooddell et al. show that a single
coinjection of NAG-MLP with potent chol-siRNAs targeting conserved
HBV sequences resulted in multilog repression of viral RNA,
proteins, and viral DNA with long duration of effect. Intravenous
coinjections, for example, of about 6 mg/kg of NAG-MLP and 6 mg/kg
of HBV specific CRISPR Cas may be envisioned for the present
invention. In the alternative, about 3 mg/kg of NAG-MLP and 3 mg/kg
of HBV specific CRISPR Cas may be delivered on day one, followed by
administration of about 2-3 mg/kg of NAG-MLP and 2-3 mg/kg of HBV
specific CRISPR Cas two weeks later.
[0696] Lin et al. (Mol Ther Nucleic Acids. 2014 Aug. 19; 3:e186.
doi: 10.1038/mtna.2014.38) designed eight gRNAs against HBV of
genotype A. With the HBV-specific gRNAs, the CRISPR-Cas9 system
significantly reduced the production of HBV core and surface
proteins in Huh-7 cells transfected with an HBV-expression vector.
Among eight screened gRNAs, two effective ones were identified. One
gRNA targeting the conserved HBV sequence acted against different
genotypes. Using a hydrodynamics-HBV persistence mouse model, Lin
et al. further demonstrated that this system could cleave the
intrahepatic HBV genome-containing plasmid and facilitate its
clearance in vivo, resulting in reduction of serum surface antigen
levels. These data suggest that the CRISPR-Cas9 system could
disrupt the HBV-expressing templates both in vitro and in vivo,
indicating its potential in eradicating persistent HBV
infection.
[0697] Dong et al. (Antiviral Res. 2015 June; 118:110-7. doi:
10.1016/j.antiviral.2015.03.015. Epub 2015 Apr. 3) used the
CRISPR-Cas9 system to target the HBV genome and efficiently inhibit
HBV infection. Dong et al. synthesized four single-guide RNAs
(guide RNAs) targeting the conserved regions of HBV. The expression
of these guide RNAS with Cas9 reduced the viral production in Huh7
cells as well as in HBV-replication cell HepG2.2.15. Dong et al.
further demonstrated that CRISPR-Cas9 direct cleavage and
cleavage-mediated mutagenesis occurred in HBV cccDNA of transfected
cells. In the mouse model carrying HBV cccDNA, injection of guide
RNA-Cas9 plasmids via rapid tail vein resulted in the low level of
cccDNA and HBV protein.
[0698] Liu et al. (J Gen Virol. 2015 August; 96(8):2252-61. doi:
10.1099/vir.0.000159. Epub 2015 Apr. 22) designed eight guide RNAs
(gRNAs) that targeted the conserved regions of different HBV
genotypes, which could significantly inhibit HBV replication both
in vitro and in vivo to investigate the possibility of using the
CRISPR-Cas9 system to disrupt the HBV DNA templates. The
HBV-specific gRNA/Cas9 system could inhibit the replication of HBV
of different genotypes in cells, and the viral DNA was
significantly reduced by a single gRNA/Cas9 system and cleared by a
combination of different gRNA/Cas9 systems.
[0699] Wang et al. (World J Gastroenterol. 2015 Aug. 28;
21(32):9554-65. doi: 10.3748/wjg.v21.i32.9554) designed 15 gRNAs
against HBV of genotypes A-D. Eleven combinations of two above
gRNAs (dual-gRNAs) covering the regulatory region of HBV were
chosen. The efficiency of each gRNA and 11 dual-gRNAs on the
suppression of HBV (genotypes A-D) replication was examined by the
measurement of HBV surface antigen (HBsAg) or e antigen (HBeAg) in
the culture supernatant. The destruction of HBV-expressing vector
was examined in HuH7 cells co-transfected with dual-gRNAs and
HBV-expressing vector using polymerase chain reaction (PCR) and
sequencing method, and the destruction of cccDNA was examined in
HepAD38 cells using KCl precipitation, plasmid-safe ATP-dependent
DNase (PSAD) digestion, rolling circle amplification and
quantitative PCR combined method. The cytotoxicity of these gRNAs
was assessed by a mitochondrial tetrazolium assay. All of gRNAs
could significantly reduce HBsAg or HBeAg production in the culture
supernatant, which was dependent on the region in which gRNA
against. All of dual gRNAs could efficiently suppress HBsAg and/or
HBeAg production for HBV of genotypes A-D, and the efficacy of dual
gRNAs in suppressing HBsAg and/or HBeAg production was
significantly increased when compared to the single gRNA used
alone. Furthermore, by PCR direct sequencing Applicants confirmed
that these dual gRNAs could specifically destroy HBV expressing
template by removing the fragment between the cleavage sites of the
two used gRNAs. Most importantly, gRNA-5 and gRNA-12 combination
not only could efficiently suppressing HBsAg and/or HBeAg
production, but also destroy the cccDNA reservoirs in HepAD38
cells.
[0700] Karimova et al. (Sci Rep. 2015 Sep. 3; 5:13734. doi:
10.1038/srep13734) identified cross-genotype conserved HBV
sequences in the S and X region of the HBV genome that were
targeted for specific and effective cleavage by a Cas9 nickase.
This approach disrupted not only episomal cccDNA and chromosomally
integrated HBV target sites in reporter cell lines, but also HBV
replication in chronically and de novo infected hepatoma cell
lines.
[0701] One of skill in the art may utilize the above studies of,
for example, Lin et al. (Mol Ther Nucleic Acids. 2014 Aug. 19;
3:e186. doi: 10.1038/mtna.2014.38), Dong et al. (Antiviral Res.
2015 June; 118:110-7. doi: 10.1016/j.antiviral.2015.03.015. Epub
2015 Apr. 3), Liu et al. (J Gen Virol. 2015 August; 96(8):2252-61.
doi: 10.1099/vir.0.000159. Epub 2015 Apr. 22), Wang et al. (World J
Gastroenterol. 2015 Aug. 28; 21(32):9554-65. doi:
10.3748/wjg.v21.i32.9554) and Karimova et al. (Sci Rep. 2015 Sep.
3; 5:13734. doi: 10.1038/srep13734) for targeting HBV with the
CRISPR Cas system of the present invention.
[0702] The present invention may also be applied to treat
pathogens, e.g. bacterial, fungal and parasitic pathogens. Most
research efforts have focused on developing new antibiotics, which
once developed, would nevertheless be subject to the same problems
of drug resistance. The invention provides novel CRISPR-based
alternatives which overcome those difficulties. Furthermore, unlike
existing antibiotics, CRISPR-based treatments can be made pathogen
specific, inducing bacterial cell death of a target pathogen while
avoiding beneficial bacteria.
[0703] Jiang et al. ("RNA-guided editing of bacterial genomes using
CRISPR-Cas systems," Nature Biotechnology vol. 31, p. 233-9, March
2013) used a CRISPR-Cas9 system to mutate or kill S. pneumoniae and
E. coli. The work, which introduced precise mutations into the
genomes, relied on dual-RNA:Cas9-directed cleavage at the targeted
genomic site to kill unmutated cells and circumvented the need for
selectable markers or counter-selection systems. CRISPR systems
have be used to reverse antibiotic resistance and eliminate the
transfer of resistance between strains. Bickard et al. showed that
Cas9, reprogrammed to target virulence genes, kills virulent, but
not avirulent, S. aureus. Reprogramming the nuclease to target
antibiotic resistance genes destroyed staphylococcal plasmids that
harbor antibiotic resistance genes and immunized against the spread
of plasmid-borne resistance genes. (see, Bikard et al., "Exploiting
CRISPR-Cas nucleases to produce sequence-specific antimicrobials,"
Nature Biotechnology vol. 32, 1146-1150, doi:10.1038/nbt.3043,
published online 5 Oct. 2014.) Bikard showed that CRISPR-Cas9
antimicrobials function in vivo to kill S. aureus in a mouse skin
colonization model. Similarly, Yosef et al used a CRISPR system to
target genes encoding enzymes that confer resistance to
.beta.-lactam antibiotics (see Yousef et al., "Temperate and lytic
bacteriophages programmed to sensitize and kill
antibiotic-resistant bacteria," Proc. Natl. Acad. Sci. USA, vol.
112, p. 7267-7272, doi: 10.1073/pnas.1500107112 published online
May 18, 2015).
[0704] CRISPR systems can be used to edit genomes of parasites that
are resistant to other genetic approaches. For example, a
CRISPR-Cas9 system was shown to introduce double-stranded breaks
into the in the Plasmodium yoelii genome (see, Zhang et al.,
"Efficient Editing of Malaria Parasite Genome Using the CRISPR/Cas9
System," mBio. vol. 5, e01414-14, July-August 2014). Ghorbal et al.
("Genome editing in the human malaria parasite Plasmodium
falciparumusing the CRISPR-Cas9 system," Nature Biotechnology, vol.
32, p. 819-821, doi: 10.1038/nbt.2925, published online Jun. 1,
2014) modified the sequences of two genes, orc1 and kelchl3, which
have putative roles in gene silencing and emerging resistance to
artemisinin, respectively. Parasites that were altered at the
appropriate sites were recovered with very high efficiency, despite
there being no direct selection for the modification, indicating
that neutral or even deleterious mutations can be generated using
this system. CRISPR-Cas9 is also used to modify the genomes of
other pathogenic parasites, including Toxoplasma gondii (see Shen
et al., "Efficient gene disruption in diverse strains of Toxoplasma
gondii using CRISPR/CAS9," mBio vol. 5:e01114-14, 2014; and Sidik
et al., "Efficient Genome Engineering of Toxoplasma gondii Using
CRISPR/Cas9," PLoS One vol. 9, e100450, doi:
10.1371/journal.pone.0100450, published online Jun. 27, 2014).
[0705] Vyas et al. ("A Candida albicans CRISPR system permits
genetic engineering of essential genes and gene families," Science
Advances, vol. 1, e1500248, DOI: 10.1126/sciadv.1500248, Apr. 3,
2015) employed a CRISPR system to overcome long-standing obstacles
to genetic engineering in C. albicans and efficiently mutate in a
single experiment both copies of several different genes. In an
organism where several mechanisms contribute to drug resistance,
Vyas produced homozygous double mutants that no longer displayed
the hyper-resistance to fluconazole or cycloheximide displayed by
the parental clinical isolate Can90. Vyas also obtained homozygous
loss-of-function mutations in essential genes of C. albicans by
creating conditional alleles. Null alleles of DCR1, which is
required for ribosomal RNA processing, are lethal at low
temperature but viable at high temperature. Vyas used a repair
template that introduced a nonsense mutation and isolated dcr1/dcr1
mutants that failed to grow at 16.degree. C.
Treating Diseases with Genetic or Epigenetic Aspects
[0706] The CRISPR-Cas systems of the present invention can be used
to correct genetic mutations that were previously attempted with
limited success using TALEN and ZFN and have been identified as
potential targets for Cas9 systems, including as in published
applications of Editas Medicine describing methods to use Cas9
systems to target loci to therapeutically address diseases with
gene therapy, including, WO 2015/048577 CRISPR-RELATED METHODS AND
COMPOSITIONS of Gluckmann et al.; WO 2015/070083 CRISPR-RELATED
METHODS AND COMPOSITIONS WITH GOVERNING gRNAS of Glucksmann et
al.
[0707] Mention is made of WO 2015/153780 CRISPR/CAS-RELATED METHODS
AND COMPOSITIONS FOR TREATING USHER SYNDROME AND RETINITIS
PIGMENTOSA of Maeder et al. Through the teachings herein the
invention comprehends methods and materials of these documents
applied in conjunction with the teachings herein. In an aspect of
ocular and auditory gene therapy, methods and compositions for
treating Usher Syndrome and Retinis-Pigmentosa may be adapted to
the CRISPR-Cas system of the present invention (see, e.g., WO
2015/134812). In an embodiment, the WO 2015/134812 involves a
treatment or delaying the onset or progression of Usher Syndrome
type IIA (USH2A, USH11A) and retinitis pigmentosa 39 (RP39) by gene
editing, e.g., using CRISPR-Cas9 mediated methods to correct the
guanine deletion at position 2299 in the USH2A gene (e.g., replace
the deleted guanine residue at position 2299 in the USH2A gene). In
a related aspect, a mutation is targeted by cleaving with either
one or more nuclease, one or more nickase, or a combination
thereof, e.g., to induce HDR with a donor template that corrects
the point mutation (e.g., the single nucleotide, e.g., guanine,
deletion). The alteration or correction of the mutant USH2A gene
can be mediated by any mechanism. Exemplary mechanisms that can be
associated with the alteration (e.g., correction) of the mutant
HSH2A gene include, but are not limited to, non-homologous end
joining, microhomology-mediated end joining (MMEJ),
homology-directed repair (e.g., endogenous donor template
mediated), SDSA (synthesis dependent strand annealing),
single-strand annealing or single strand invasion. In an
embodiment, the method used for treating Usher Syndrome and
Retinis-Pigmentosa can include acquiring knowledge of the mutation
carried by the subject, e.g., by sequencing the appropriate portion
of the USH2A gene.
[0708] Mention is also made of WO 2015/138510 and through the
teachings herein the invention (using a CRISPR-Cas9 system)
comprehends providing a treatment or delaying the onset or
progression of Leber's Congenital Amaurosis 10 (LCA 10). LCA 10 is
caused by a mutation in the CEP290 gene, e.g., a c.2991+1655,
adenine to guanine mutation in the CEP290 gene which gives rise to
a cryptic splice site in intron 26. This is a mutation at
nucleotide 1655 of intron 26 of CEP290, e.g., an A to G mutation.
CEP290 is also known as: CT87; MKS4; POC3; rd16; BBS14; JBTS5;
LCAJO; NPHP6; SLSN6; and 3H11Ag (see, e.g., WO 2015/138510). In an
aspect of gene therapy, the invention involves introducing one or
more breaks near the site of the LCA target position (e.g.,
c.2991+1655; A to G) in at least one allele of the CEP290 gene.
Altering the LCA10 target position refers to (1) break-induced
introduction of an indel (also referred to herein as NHEJ-mediated
introduction of an indel) in close proximity to or including a
LCA10 target position (e.g., c.2991+1655A to G), or (2)
break-induced deletion (also referred to herein as NHEJ-mediated
deletion) of genomic sequence including the mutation at a LCA10
target position (e.g., c.2991+1655A to G). Both approaches give
rise to the loss or destruction of the cryptic splice site
resulting from the mutation at the LCA 10 target position.
[0709] In an aspect, the invention (using a CRISPR-Cas9 system)
comprehends providing a treatment or delaying the onset or
progression of Leber's Congenital Amaurosis 10 (LCA 10). LCA 10 is
caused by a mutation in the CEP290 gene, e.g., a c.2991+1655,
adenine to guanine mutation in the CEP290 gene which gives rise to
a cryptic splice site in intron 26. This is a mutation at
nucleotide 1655 of intron 26 of CEP290, e.g., an A to G mutation.
CEP290 is also known as: CT87; MKS4; POC3; rd16; BBS14; JBTS5;
LCAJO; NPHP6; SLSN6; and 3H11Ag (see, e.g., WO 2015/138510). In an
aspect of gene therapy, the invention involves introducing one or
more breaks near the site of the LCA target position (e.g.,
c.2991+1655; A to G) in at least one allele of the CEP290 gene.
Altering the LCA10 target position refers to (1) break-induced
introduction of an indel (also referred to herein as NHEJ-mediated
introduction of an indel) in close proximity to or including a
LCA10 target position (e.g., c.2991+1655A to G), or (2)
break-induced deletion (also referred to herein as NHEJ-mediated
deletion) of genomic sequence including the mutation at a LCA10
target position (e.g., c.2991+1655A to G). Both approaches give
rise to the loss or destruction of the cryptic splice site
resulting from the mutation at the LCA 10 target position.
[0710] Researchers are contemplating whether gene therapies could
be employed to treat a wide range of diseases. The CRISPR systems
of the present invention based on Cas9 effector protein are
envisioned for such therapeutic uses, including, but noted limited
to further exemplified targeted areas and with delivery methods as
below. Some examples of conditions or diseases that might be
usefully treated using the present system are included in the
examples of genes and references included herein and are currently
associated with those conditions are also provided there. The genes
and conditions exemplified are not exhaustive.
Treating Diseases of the Circulatory System
[0711] The present invention also contemplates delivering the
CRISPR-Cas system, specifically the novel CRISPR effector protein
systems described herein, to the blood or hematopoetic stem cells.
The plasma exosomes of Wahlgren et al. (Nucleic Acids Research,
2012, Vol. 40, No. 17 e130) were previously described and may be
utilized to deliver the CRISPR Cas system to the blood. The nucleic
acid-targeting system of the present invention is also contemplated
to treat hemoglobinopathies, such as thalassemias and sickle cell
disease. See, e.g., International Patent Publication No. WO
2013/126794 for potential targets that may be targeted by the
CRISPR Cas system of the present invention.
[0712] Drakopoulou, "Review Article, The Ongoing Challenge of
Hematopoietic Stem Cell-Based Gene Therapy for .beta.-Thalassemia,"
Stem Cells International, Volume 2011, Article ID 987980, 10 pages,
doi:10.4061/2011/987980, incorporated herein by reference along
with the documents it cites, as if set out in full, discuss
modifying HSCs using a lentivirus that delivers a gene for
.beta.-globin or .gamma.-globin. In contrast to using lentivirus,
with the knowledge in the art and the teachings in this disclosure,
the skilled person can correct HSCs as to .beta.-Thalassemia using
a CRISPR-Cas system that targets and corrects the mutation (e.g.,
with a suitable HDR template that delivers a coding sequence for
.beta.-globin or .gamma.-globin, advantageously non-sickling
.beta.-globin or .gamma.-globin); specifically, the guide RNA can
target mutation that give rise to .beta.-Thalassemia, and the HDR
can provide coding for proper expression of .beta.-globin or
.gamma.-globin. A guide RNA that targets the mutation-and-Cas
protein containing particle is contacted with HSCs carrying the
mutation. The particle also can contain a suitable HDR template to
correct the mutation for proper expression of .beta.-globin or
.gamma.-globin; or the HSC can be contacted with a second particle
or a vector that contains or delivers the HDR template. The so
contacted cells can be administered; and optionally
treated/expanded; cf. Cartier. In this regard mention is made of:
Cavazzana, "Outcomes of Gene Therapy for .beta.-Thalassemia Major
via Transplantation of Autologous Hematopoietic Stem Cells
Transduced Ex Vivo with a Lentiviral .beta..sup.A-T87Q-Globin
Vector." tif2014. org/abstractFiles/Jean %20Antoine
%20Ribeil_Abstract.pdf; Cavazzana-Calvo, "Transfusion independence
and HMGA2 activation after gene therapy of human
.beta.-thalassaemia", Nature 467, 318-322 (16 Sep. 2010)
doi:10.1038/nature09328; Nienhuis, "Development of Gene Therapy for
Thalassemia, Cold Spring Harbor Perspectives in Medicine, doi:
10.1101/cshperspect.a011833 (2012), LentiGlobin BB305, a lentiviral
vector containing an engineered .beta.-globin gene (.beta.A-T87Q);
and Xie et al., "Seamless gene correction of .beta.-thalassaemia
mutations in patient-specific iPSCs using CRISPR/Cas9 and
piggyback" Genome Research gr.173427.114 (2014)
http://www.genome.org/cgi/doi/10.1101/gr.173427.114 (Cold Spring
Harbor Laboratory Press); that is the subject of Cavazzana work
involving human .beta.-thalassaemia and the subject of the Xie
work, are all incorporated herein by reference, together with all
documents cited therein or associated therewith. In the instant
invention, the HDR template can provide for the HSC to express an
engineered .beta.-globin gene (e.g., .beta.A-T87Q), or
.beta.-globin as in Xie.
[0713] Xu et al. (Sci Rep. 2015 Jul. 9; 5:12065. doi:
10.1038/srep12065) have designed TALENs and CRISPR-Cas9 to directly
target the intron2 mutation site IVS2-654 in the globin gene. Xu et
al. observed different frequencies of double-strand breaks (DSBs)
at IVS2-654 loci using TALENs and CRISPR-Cas9, and TALENs mediated
a higher homologous gene targeting efficiency compared to
CRISPR-Cas9 when combined with the piggyBac transposon donor. In
addition, more obvious off-target events were observed for
CRISPR-Cas9 compared to TALENs. Finally, TALENs-corrected iPSC
clones were selected for erythroblast differentiation using the OP9
co-culture system and detected relatively higher transcription of
HBB than the uncorrected cells.
[0714] Song et al. (Stem Cells Dev. 2015 May 1; 24(9):1053-65. doi:
10.1089/scd.2014.0347. Epub 2015 Feb. 5) used CRISPR/Cas9 to
correct .beta.-Thal iPSCs; gene-corrected cells exhibit normal
karyotypes and full pluripotency as human embryonic stem cells
(hESCs) showed no off-targeting effects. Then, Song et al.
evaluated the differentiation efficiency of the gene-corrected
.beta.-Thal iPSCs. Song et al. found that during hematopoietic
differentiation, gene-corrected .beta.-Thal iPSCs showed an
increased embryoid body ratio and various hematopoietic progenitor
cell percentages. More importantly, the gene-corrected .beta.-Thal
iPSC lines restored HBB expression and reduced reactive oxygen
species production compared with the uncorrected group. Song et
al.'s study suggested that hematopoietic differentiation efficiency
of .beta.-Thal iPSCs was greatly improved once corrected by the
CRISPR-Cas9 system. Similar methods may be performed utilizing the
CRISPR-Cas systems described herein, e.g. systems comprising Cas9
effector proteins.
[0715] Mention is made of WO 2015/148860, through the teachings
herein the invention comprehends methods and materials of these
documents applied in conjunction with the teachings herein. In an
aspect of blood-related disease gene therapy, methods and
compositions for treating beta thalassemia may be adapted to the
CRISPR-Cas system of the present invention (see, e.g., WO
2015/148860). In an embodiment, WO 2015/148860 involves the
treatment or prevention of beta thalassemia, or its symptoms, e.g.,
by altering the gene for B-cell CLL/lymphoma 11A (BCL11A). The
BCL11A gene is also known as B-cell CLL/lymphoma 11A, BCL11A-L,
BCL11A-S, BCL11AXL, CTIP 1, HBFQTL5 and ZNF. BCL11A encodes a
zinc-finger protein that is involved in the regulation of globin
gene expression. By altering the BCL11A gene (e.g., one or both
alleles of the BCL11A gene), the levels of gamma globin can be
increased. Gamma globin can replace beta globin in the hemoglobin
complex and effectively carry oxygen to tissues, thereby
ameliorating beta thalassemia disease phenotypes.
[0716] Sickle cell anemia is an autosomal recessive genetic disease
in which red blood cells become sickle-shaped. It is caused by a
single base substitution in the 3-globin gene, which is located on
the short arm of chromosome 11. As a result, valine is produced
instead of glutamic acid causing the production of sickle
hemoglobin (HbS). This results in the formation of a distorted
shape of the erythrocytes. Due to this abnormal shape, small blood
vessels can be blocked, causing serious damage to the bone, spleen
and skin tissues. This may lead to episodes of pain, frequent
infections, hand-foot syndrome or even multiple organ failure. The
distorted erythrocytes are also more susceptible to hemolysis,
which leads to serious anemia. As in the case of
.beta.-thalassaemia, sickle cell anemia can be corrected by
modifying HSCs with the CRISPR-Cas system. The system allows the
specific editing of the cell's genome by cutting its DNA and then
letting it repair itself. The Cas9 protein is inserted and directed
by a RNA guide to the mutated point and then it cuts the DNA at
that point. Simultaneously, a healthy version of the sequence is
inserted. This sequence is used by the cell's own repair system to
fix the induced cut. In this way, the CRISPR-Cas allows the
correction of the mutation in the previously obtained stem cells.
With the knowledge in the art and the teachings in this disclosure,
the skilled person can correct HSCs as to sickle cell anemia using
a CRISPR-Cas system that targets and corrects the mutation (e.g.,
with a suitable HDR template that delivers a coding sequence for
.beta.-globin, advantageously non-sickling .beta.-globin);
specifically, the guide RNA can target mutation that give rise to
sickle cell anemia, and the HDR can provide coding for proper
expression of .beta.-globin. An guide RNA that targets the
mutation-and-Cas protein containing particle is contacted with HSCs
carrying the mutation. The particle also can contain a suitable HDR
template to correct the mutation for proper expression of
.beta.-globin; or the HSC can be contacted with a second particle
or a vector that contains or delivers the HDR template. The so
contacted cells can be administered; and optionally
treated/expanded; cf. Cartier. The HDR template can provide for the
HSC to express an engineered .beta.-globin gene (e.g.,
.beta.A-T87Q), or .beta.-globin as in Xie.
[0717] Mention is also made of WO 2015/148863 and through the
teachings herein the invention comprehends methods and materials of
these documents which may be adapted to the CRISPR-Cas system of
the present invention. In an aspect of treating and preventing
sickle cell disease, which is an inherited hematologic disease, WO
2015/148863 comprehends altering the BCL11A gene. By altering the
BCL11A gene (e.g., one or both alleles of the BCL11A gene), the
levels of gamma globin can be increased. Gamma globin can replace
beta globin in the hemoglobin complex and effectively carry oxygen
to tissues, thereby ameliorating sickle cell disease
phenotypes.
[0718] Williams, "Broadening the Indications for Hematopoietic Stem
Cell Genetic Therapies," Cell Stem Cell 13:263-264 (2013),
incorporated herein by reference along with the documents it cites,
as if set out in full, report lentivirus-mediated gene transfer
into HSC/P cells from patients with the lysosomal storage disease
metachromatic leukodystrophy disease (MLD), a genetic disease
caused by deficiency of arylsulfatase A (ARSA), resulting in nerve
demyelination; and lentivirus-mediated gene transfer into HSCs of
patients with Wiskott-Aldrich syndrome (WAS) (patients with
defective WAS protein, an effector of the small GTPase CDC42 that
regulates cytoskeletal function in blood cell lineages and thus
suffer from immune deficiency with recurrent infections, autoimmune
symptoms, and thrombocytopenia with abnormally small and
dysfunctional platelets leading to excessive bleeding and an
increased risk of leukemia and lymphoma). In contrast to using
lentivirus, with the knowledge in the art and the teachings in this
disclosure, the skilled person can correct HSCs as to MLD
(deficiency of arylsulfatase A (ARSA)) using a CRISPR-Cas system
that targets and corrects the mutation (deficiency of arylsulfatase
A (ARSA)) (e.g., with a suitable HDR template that delivers a
coding sequence for ARSA); specifically, the guide RNA can target
mutation that gives rise to MLD (deficient ARSA), and the HDR can
provide coding for proper expression of ARSA. A guide RNA that
targets the mutation-and-Cas protein containing particle is
contacted with HSCs carrying the mutation. The particle also can
contain a suitable HDR template to correct the mutation for proper
expression of ARSA; or the HSC can be contacted with a second
particle or a vector that contains or delivers the HDR template.
The so contacted cells can be administered; and optionally
treated/expanded; cf. Cartier. In contrast to using lentivirus,
with the knowledge in the art and the teachings in this disclosure,
the skilled person can correct HSCs as to WAS using a CRISPR-Cas
system that targets and corrects the mutation (deficiency of WAS
protein) (e.g., with a suitable HDR template that delivers a coding
sequence for WAS protein); specifically, the guide RNA can target
mutation that gives rise to WAS (deficient WAS protein), and the
HDR can provide coding for proper expression of WAS protein. A
guide RNA that targets the mutation-and-Cas9 protein containing
particle is contacted with HSCs carrying the mutation. The particle
also can contain a suitable HDR template to correct the mutation
for proper expression of WAS protein; or the HSC can be contacted
with a second particle or a vector that contains or delivers the
HDR template. The so contacted cells can be administered; and
optionally treated/expanded; cf. Cartier.
[0719] In an aspect of the invention, methods and compositions
which involve editing a target nucleic acid sequence, or modulating
expression of a target nucleic acid sequence, and applications
thereof in connection with cancer immunotherapy are comprehended by
adapting the CRISPR-Cas system of the present invention. Reference
is made to the application of gene therapy in WO 2015/161276 which
involves methods and compositions which can be used to affect
T-cell proliferation, survival and/or function by altering one or
more T-cell expressed genes, e.g., one or more of FAS, BID, CTLA4,
PDCD1, CBLB, PTPN6, TRAC and/or TRBC genes. In a related aspect,
T-cell proliferation can be affected by altering one or more T-cell
expressed genes, e.g., the CBLB and/or PTPN6 gene, FAS and/or BID
gene, CTLA4 and/or PDCDI and/or TRAC and/or TRBC gene.
[0720] Chimeric antigen receptor (CAR)19 T-cells exhibit
anti-leukemic effects in patient malignancies. However, leukemia
patients often do not have enough T-cells to collect, meaning that
treatment must involve modified T cells from donors. Accordingly,
there is interest in establishing a bank of donor T-cells. Qasim et
al. ("First Clinical Application of Talen Engineered Universal
CAR19 T Cells in B-ALL" ASH 57th Annual Meeting and Exposition,
Dec. 5-8, 2015, Abstract 2046
(https://ash.confex.com/ash/2015/webprogram/Paper81653.html
published online November 2015) discusses modifying CAR19 T cells
to eliminate the risk of graft-versus-host disease through the
disruption of T-cell receptor expression and CD52 targeting.
Furthermore, CD52 cells were targeted such that they became
insensitive to Alemtuzumab, and thus allowed Alemtuzumab to prevent
host-mediated rejection of human leukocyte antigen (HLA) mismatched
CAR19 T-cells. Investigators used third generation
self-inactivating lentiviral vector encoding a 4g7 CAR19 (CD19
scFv-4-1BB-CD3.zeta.) linked to RQR8, then electroporated cells
with two pairs of TALEN mRNA for multiplex targeting for both the
T-cell receptor (TCR) alpha constant chain locus and the CD52 gene
locus. Cells which were still expressing TCR following ex vivo
expansion were depleted using CliniMacs .alpha./.beta. TCR
depletion, yielding a T-cell product (UCART19) with <1% TCR
expression, 85% of which expressed CAR19, and 64% becoming CD52
negative. The modified CAR19 T cells were administered to treat a
patient's relapsed acute lymphoblastic leukemia. The teachings
provided herein provide effective methods for modifying cells, for
example to remove or modulate CD52 or other targets, thus can be
used in conjunction with modification of administration of T cells
or other cells to patients to treat malignancies.
[0721] Watts, "Hematopoietic Stem Cell Expansion and Gene Therapy"
Cytotherapy 13(10):1164-1171. doi:10.3109/14653249.2011.620748
(2011), incorporated herein by reference along with the documents
it cites, as if set out in full, discusses hematopoietic stem cell
(HSC) gene therapy, e.g., virus-mediated HSC gene therapy, as an
highly attractive treatment option for many disorders including
hematologic conditions, immunodeficiencies including HIV/AIDS, and
other genetic disorders like lysosomal storage diseases, including
SCID-X1, ADA-SCID, .beta.-thalassemia, X-linked CGD,
Wiskott-Aldrich syndrome, Fanconi anemia, adrenoleukodystrophy
(ALD), and metachromatic leukodystrophy (MLD).
[0722] US Patent Publication Nos. 20110225664, 20110091441,
20100229252, 20090271881 and 20090222937 assigned to Cellectis,
relates to CREI variants, wherein at least one of the two I-CreI
monomers has at least two substitutions, one in each of the two
functional subdomains of the LAGLIDADG core domain ("LAGLIDADG"
disclosed as SEQ ID NO: 49) situated respectively from positions 26
to 40 and 44 to 77 of I-CreI, said variant being able to cleave a
DNA target sequence from the human interleukin-2 receptor gamma
chain (IL2RG) gene also named common cytokine receptor gamma chain
gene or gamma C gene. The target sequences identified in US Patent
Publication Nos. 20110225664, 20110091441, 20100229252, 20090271881
and 20090222937 may be utilized for the nucleic acid-targeting
system of the present invention.
[0723] Severe Combined Immune Deficiency (SCID) results from a
defect in lymphocytes T maturation, always associated with a
functional defect in lymphocytes B (Cavazzana-Calvo et al., Annu.
Rev. Med., 2005, 56, 585-602; Fischer et al., Immunol. Rev., 2005,
203, 98-109). Overall incidence is estimated to 1 in 75 000 births.
Patients with untreated SCID are subject to multiple opportunist
micro-organism infections, and do generally not live beyond one
year. SCID can be treated by allogenic hematopoietic stem cell
transfer, from a familial donor. Histocompatibility with the donor
can vary widely. In the case of Adenosine Deaminase (ADA)
deficiency, one of the SCID forms, patients can be treated by
injection of recombinant Adenosine Deaminase enzyme.
[0724] Since the ADA gene has been shown to be mutated in SCID
patients (Giblett et al., Lancet, 1972, 2, 1067-1069), several
other genes involved in SCID have been identified (Cavazzana-Calvo
et al., Annu. Rev. Med., 2005, 56, 585-602; Fischer et al.,
Immunol. Rev., 2005, 203, 98-109). There are four major causes for
SCID: (i) the most frequent form of SCID, SCID-X1 (X-linked SCID or
X-SCID), is caused by mutation in the IL2RG gene, resulting in the
absence of mature T lymphocytes and NK cells. IL2RG encodes the
gamma C protein (Noguchi, et al., Cell, 1993, 73, 147-157), a
common component of at least five interleukin receptor complexes.
These receptors activate several targets through the JAK3 kinase
(Macchi et al., Nature, 1995, 377, 65-68), which inactivation
results in the same syndrome as gamma C inactivation; (ii) mutation
in the ADA gene results in a defect in purine metabolism that is
lethal for lymphocyte precursors, which in turn results in the
quasi absence of B, T and NK cells; (iii) V(D)J recombination is an
essential step in the maturation of immunoglobulins and T
lymphocytes receptors (TCRs). Mutations in Recombination Activating
Gene 1 and 2 (RAG1 and RAG2) and Artemis, three genes involved in
this process, result in the absence of mature T and B lymphocytes;
and (iv) Mutations in other genes such as CD45, involved in T cell
specific signaling have also been reported, although they represent
a minority of cases (Cavazzana-Calvo et al., Annu. Rev. Med., 2005,
56, 585-602; Fischer et al., Immunol. Rev., 2005, 203, 98-109).
Since when their genetic bases have been identified, the different
SCID forms have become a paradigm for gene therapy approaches
(Fischer et al., Immunol. Rev., 2005, 203, 98-109) for two major
reasons. First, as in all blood diseases, an ex vivo treatment can
be envisioned. Hematopoietic Stem Cells (HSCs) can be recovered
from bone marrow, and keep their pluripotent properties for a few
cell divisions. Therefore, they can be treated in vitro, and then
reinjected into the patient, where they repopulate the bone marrow.
Second, since the maturation of lymphocytes is impaired in SCID
patients, corrected cells have a selective advantage. Therefore, a
small number of corrected cells can restore a functional immune
system. This hypothesis was validated several times by (i) the
partial restoration of immune functions associated with the
reversion of mutations in SCID patients (Hirschhorn et al., Nat.
Genet., 1996, 13, 290-295; Stephan et al., N. Engl. J. Med., 1996,
335, 1563-1567; Bousso et al., Proc. Natl., Acad. Sci. USA, 2000,
97, 274-278; Wada et al., Proc. Natl. Acad. Sci. USA, 2001, 98,
8697-8702; Nishikomori et al., Blood, 2004, 103, 4565-4572), (ii)
the correction of SCID-X1 deficiencies in vitro in hematopoietic
cells (Candotti et al., Blood, 1996, 87, 3097-3102; Cavazzana-Calvo
et al., Blood, 1996, Blood, 88, 3901-3909; Taylor et al., Blood,
1996, 87, 3103-3107; Hacein-Bey et al., Blood, 1998, 92,
4090-4097), (iii) the correction of SCID-X1 (Soudais et al., Blood,
2000, 95, 3071-3077; Tsai et al., Blood, 2002, 100, 72-79), JAK-3
(Bunting et al., Nat. Med., 1998, 4, 58-64; Bunting et al., Hum.
Gene Ther., 2000, 11, 2353-2364) and RAG2 (Yates et al., Blood,
2002, 100, 3942-3949) deficiencies in vivo in animal models and
(iv) by the result of gene therapy clinical trials (Cavazzana-Calvo
et al., Science, 2000, 288, 669-672; Aiuti et al., Nat. Med., 2002;
8, 423-425; Gaspar et al., Lancet, 2004, 364, 2181-2187).
[0725] US Patent Publication No. 20110182867 assigned to the
Children's Medical Center Corporation and the President and Fellows
of Harvard College relates to methods and uses of modulating fetal
hemoglobin expression (HbF) in a hematopoietic progenitor cells via
inhibitors of BCL11A expression or activity, such as RNAi and
antibodies. The targets disclosed in US Patent Publication No.
20110182867, such as BCL11A, may be targeted by the CRISPR Cas
system of the present invention for modulating fetal hemoglobin
expression. See also Bauer et al. (Science 11 Oct. 2013: Vol. 342
no. 6155 pp. 253-257) and Xu et al. (Science 18 Nov. 2011: Vol. 334
no. 6058 pp. 993-996) for additional BCL11A targets.
[0726] With the knowledge in the art and the teachings in this
disclosure, the skilled person can correct HSCs as to a genetic
hematologic disorder, e.g., .beta.-Thalassemia, Hemophilia, or a
genetic lysosomal storage disease.
Treating Disease of the Brain, Central Nervous and Immune
Systems
[0727] The present invention also contemplates delivering the
CRISPR-Cas system to the brain or neurons. For example, RNA
interference (RNAi) offers therapeutic potential for this disorder
by reducing the expression of HTT, the disease-causing gene of
Huntington's disease (see, e.g., McBride et al., Molecular Therapy
vol. 19 no. 12 Dec. 2011, pp. 2152-2162), therefore Applicant
postulates that it may be used/and or adapted to the CRISPR-Cas
system. The CRISPR-Cas system may be generated using an algorithm
to reduce the off-targeting potential of antisense sequences. The
CRISPR-Cas sequences may target either a sequence in exon 52 of
mouse, rhesus or human huntingtin and expressed in a viral vector,
such as AAV. Animals, including humans, may be injected with about
three microinjections per hemisphere (six injections total): the
first 1 mm rostral to the anterior commissure (12 .mu.l) and the
two remaining injections (12 .mu.l and 10 .mu.l, respectively)
spaced 3 and 6 mm caudal to the first injection with 1e12 vg/ml of
AAV at a rate of about 1 .mu.l/minute, and the needle was left in
place for an additional 5 minutes to allow the injectate to diffuse
from the needle tip.
[0728] DiFiglia et al. (PNAS, Oct. 23, 2007, vol. 104, no. 43,
17204-17209) observed that single administration into the adult
striatum of an siRNA targeting Htt can silence mutant Htt,
attenuate neuronal pathology, and delay the abnormal behavioral
phenotype observed in a rapid-onset, viral transgenic mouse model
of HD. DiFiglia injected mice intrastriatally with 2 .mu.l of
Cy3-labeled cc-siRNA-Htt or unconjugated siRNA-Htt at 10 .mu.M. A
similar dosage of CRISPR Cas targeted to Htt may be contemplated
for humans in the present invention, for example, about 5-10 ml of
10 .mu.M CRISPR Cas targeted to Htt may be injected
intrastriatally.
[0729] In another example, Boudreau et al. (Molecular Therapy vol.
17 no. 6 Jun. 2009) injects 5 .mu.l of recombinant AAV serotype 2/1
vectors expressing htt-specific RNAi virus (at 4.times.10.sup.12
viral genomes/ml) into the striatum. A similar dosage of CRISPR Cas
targeted to Htt may be contemplated for humans in the present
invention, for example, about 10-20 ml of 4.times.10.sup.12 viral
genomes/ml) CRISPR Cas targeted to Htt may be injected
intrastriatally.
[0730] In another example, a CRISPR Cas targeted to HTT may be
administered continuously (see, e.g., Yu et al., Cell 150, 895-908,
Aug. 31, 2012). Yu et al. utilizes osmotic pumps delivering 0.25
ml/hr (Model 2004) to deliver 300 mg/day of ss-siRNA or
phosphate-buffered saline (PBS) (Sigma Aldrich) for 28 days, and
pumps designed to deliver 0.5 .mu.l/hr (Model 2002) were used to
deliver 75 mg/day of the positive control MOE ASO for 14 days.
Pumps (Durect Corporation) were filled with ss-siRNA or MOE diluted
in sterile PBS and then incubated at 37 C for 24 or 48 (Model 2004)
hours prior to implantation. Mice were anesthetized with 2.5%
isofluorane, and a midline incision was made at the base of the
skull. Using stereotaxic guides, a cannula was implanted into the
right lateral ventricle and secured with Loctite adhesive. A
catheter attached to an Alzet osmotic mini pump was attached to the
cannula, and the pump was placed subcutaneously in the midscapular
area. The incision was closed with 5.0 nylon sutures. A similar
dosage of CRISPR Cas targeted to Htt may be contemplated for humans
in the present invention, for example, about 500 to 1000 g/day
CRISPR Cas targeted to Htt may be administered.
[0731] In another example of continuous infusion, Stiles et al.
(Experimental Neurology 233 (2012) 463-471) implanted an
intraparenchymal catheter with a titanium needle tip into the right
putamen. The catheter was connected to a SynchroMed.RTM. II Pump
(Medtronic Neurological, Minneapolis, Minn.) subcutaneously
implanted in the abdomen. After a 7 day infusion of phosphate
buffered saline at 6 .mu.L/day, pumps were re-filled with test
article and programmed for continuous delivery for 7 days. About
2.3 to 11.52 mg/d of siRNA were infused at varying infusion rates
of about 0.1 to 0.5 .mu.L/min. A similar dosage of CRISPR Cas
targeted to Htt may be contemplated for humans in the present
invention, for example, about 20 to 200 mg/day CRISPR Cas targeted
to Htt may be administered. In another example, the methods of US
Patent Publication No. 20130253040 assigned to Sangamo may also be
also be adapted from TALES to the nucleic acid-targeting system of
the present invention for treating Huntington's Disease.
[0732] A further aspect of the invention relates to utilizing the
CRISPR-Cas system for correcting defects in the EMP2A and EMP2B
genes that have been identified to be associated with Lafora
disease. Lafora disease is an autosomal recessive condition which
is characterized by progressive myoclonus epilepsy which may start
as epileptic seizures in adolescence. A few cases of the disease
may be caused by mutations in genes yet to be identified. The
disease causes seizures, muscle spasms, difficulty walking,
dementia, and eventually death. There is currently no therapy that
has proven effective against disease progression. Other genetic
abnormalities associated with epilepsy may also be targeted by the
CRISPR-Cas system and the underlying genetics is further described
in Genetics of Epilepsy and Genetic Epilepsies, edited by Giuliano
Avanzini, Jeffrey L. Noebels, Mariani Foundation Paediatric
Neurology:20; 2009).
[0733] The methods of US Patent Publication No. 20110158957
assigned to Sangamo BioSciences, Inc. involved in inactivating T
cell receptor (TCR) genes may also be modified to the CRISPR Cas
system of the present invention. In another example, the methods of
US Patent Publication No. 20100311124 assigned to Sangamo
BioSciences, Inc. and US Patent Publication No. 20110225664
assigned to Cellectis, which are both involved in inactivating
glutamine synthetase gene expression genes may also be modified to
the CRISPR Cas system of the present invention.
Treating Hearing Diseases
[0734] The present invention also contemplates delivering the
CRISPR-Cas system to one or both ears.
[0735] Researchers are looking into whether gene therapy could be
used to aid current deafness treatments--namely, cochlear implants.
Deafness is often caused by lost or damaged hair cells that cannot
relay signals to auditory neurons. In such cases, cochlear implants
may be used to respond to sound and transmit electrical signals to
the nerve cells. But these neurons often degenerate and retract
from the cochlea as fewer growth factors are released by impaired
hair cells.
[0736] US patent application 20120328580 describes injection of a
pharmaceutical composition into the ear (e.g., auricular
administration), such as into the luminae of the cochlea (e.g., the
Scala media, Sc vestibulae, and Sc tympani), e.g., using a syringe,
e.g., a single-dose syringe. For example, one or more of the
compounds described herein can be administered by intratympanic
injection (e.g., into the middle ear), and/or injections into the
outer, middle, and/or inner ear. Such methods are routinely used in
the art, for example, for the administration of steroids and
antibiotics into human ears. Injection can be, for example, through
the round window of the ear or through the cochlear capsule. Other
inner ear administration methods are known in the art (see, e.g.,
Salt and Plontke, Drug Discovery Today, 10:1299-1306, 2005).
[0737] In another mode of administration, the pharmaceutical
composition can be administered in situ, via a catheter or pump. A
catheter or pump can, for example, direct a pharmaceutical
composition into the cochlear luminae or the round window of the
ear and/or the lumen of the colon. Exemplary drug delivery
apparatus and methods suitable for administering one or more of the
compounds described herein into an ear, e.g., a human ear, are
described by McKenna et al., (U.S. Publication No. 2006/0030837)
and Jacobsen et al., (U.S. Pat. No. 7,206,639). In some
embodiments, a catheter or pump can be positioned, e.g., in the ear
(e.g., the outer, middle, and/or inner ear) of a patient during a
surgical procedure. In some embodiments, a catheter or pump can be
positioned, e.g., in the ear (e.g., the outer, middle, and/or inner
ear) of a patient without the need for a surgical procedure.
[0738] Alternatively or in addition, one or more of the compounds
described herein can be administered in combination with a
mechanical device such as a cochlear implant or a hearing aid,
which is worn in the outer ear. An exemplary cochlear implant that
is suitable for use with the present invention is described by Edge
et al., (U.S. Publication No. 2007/0093878).
[0739] In some embodiments, the modes of administration described
above may be combined in any order and can be simultaneous or
interspersed.
[0740] Alternatively or in addition, the present invention may be
administered according to any of the Food and Drug Administration
approved methods, for example, as described in CDER Data Standards
Manual, version number 004 (which is available at
fda.give/cder/dsm/DRG/drg00301.htm).
[0741] In general, the cell therapy methods described in US patent
application 20120328580 can be used to promote complete or partial
differentiation of a cell to or towards a mature cell type of the
inner ear (e.g., a hair cell) in vitro. Cells resulting from such
methods can then be transplanted or implanted into a patient in
need of such treatment. The cell culture methods required to
practice these methods, including methods for identifying and
selecting suitable cell types, methods for promoting complete or
partial differentiation of selected cells, methods for identifying
complete or partially differentiated cell types, and methods for
implanting complete or partially differentiated cells are described
below.
[0742] Cells suitable for use in the present invention include, but
are not limited to, cells that are capable of differentiating
completely or partially into a mature cell of the inner ear, e.g.,
a hair cell (e.g., an inner and/or outer hair cell), when
contacted, e.g., in vitro, with one or more of the compounds
described herein. Exemplary cells that are capable of
differentiating into a hair cell include, but are not limited to
stem cells (e.g., inner ear stem cells, adult stem cells, bone
marrow derived stem cells, embryonic stem cells, mesenchymal stem
cells, skin stem cells, iPS cells, and fat derived stem cells),
progenitor cells (e.g., inner ear progenitor cells), support cells
(e.g., Deiters' cells, pillar cells, inner phalangeal cells, tectal
cells and Hensen's cells), and/or germ cells. The use of stem cells
for the replacement of inner ear sensory cells is described in Li
et al., (U.S. Publication No. 2005/0287127) and Li et al., (U.S.
patent Ser. No. 11/953,797). The use of bone marrow derived stem
cells for the replacement of inner ear sensory cells is described
in Edge et al., PCT/US2007/084654. iPS cells are described, e.g.,
at Takahashi et al., Cell, Volume 131, Issue 5, Pages 861-872
(2007); Takahashi and Yamanaka, Cell 126, 663-76 (2006); Okita et
al., Nature 448, 260-262 (2007); Yu, J. et al., Science
318(5858):1917-1920 (2007); Nakagawa et al., Nat. Biotechnol.
26:101-106 (2008); and Zaehres and Scholer, Cell 131(5):834-835
(2007). Such suitable cells can be identified by analyzing (e.g.,
qualitatively or quantitatively) the presence of one or more tissue
specific genes. For example, gene expression can be detected by
detecting the protein product of one or more tissue-specific genes.
Protein detection techniques involve staining proteins (e.g., using
cell extracts or whole cells) using antibodies against the
appropriate antigen. In this case, the appropriate antigen is the
protein product of the tissue-specific gene expression. Although,
in principle, a first antibody (i.e., the antibody that binds the
antigen) can be labeled, it is more common (and improves the
visualization) to use a second antibody directed against the first
(e.g., an anti-IgG). This second antibody is conjugated either with
fluorochromes, or appropriate enzymes for colorimetric reactions,
or gold beads (for electron microscopy), or with the biotin-avidin
system, so that the location of the primary antibody, and thus the
antigen, can be recognized.
[0743] The CRISPR Cas molecules of the present invention may be
delivered to the ear by direct application of pharmaceutical
composition to the outer ear, with compositions modified from US
Published application, 20110142917. In some embodiments the
pharmaceutical composition is applied to the ear canal. Delivery to
the ear may also be referred to as aural or otic delivery.
[0744] In some embodiments the RNA molecules of the invention are
delivered in liposome or lipofectin formulations and the like and
can be prepared by methods well known to those skilled in the art.
Such methods are described, for example, in U.S. Pat. Nos.
5,593,972, 5,589,466, and 5,580,859, which are herein incorporated
by reference.
[0745] Delivery systems aimed specifically at the enhanced and
improved delivery of siRNA into mammalian cells have been
developed, (see, for example, Shen et al FEBS Let. 2003,
539:111-114; Xia et al., Nat. Biotech. 2002, 20:1006-1010; Reich et
al., Mol. Vision. 2003, 9: 210-216; Sorensen et al., J. Mol. Biol.
2003, 327: 761-766; Lewis et al., Nat. Gen. 2002, 32: 107-108 and
Simeoni et al., NAR 2003, 31, 11: 2717-2724) and may be applied to
the present invention. siRNA has recently been successfully used
for inhibition of gene expression in primates (see for example
Tolentino et al., Retina 24(4):660 which may also be applied to the
present invention).
[0746] Qi et al. discloses methods for efficient siRNA transfection
to the inner ear through the intact round window by a novel
proteidic delivery technology which may be applied to the nucleic
acid-targeting system of the present invention (see, e.g., Qi et
al., Gene Therapy (2013), 1-9). In particular, a TAT double
stranded RNA-binding domains (TAT-DRBDs), which can transfect
Cy3-labeled siRNA into cells of the inner ear, including the inner
and outer hair cells, crista ampullaris, macula utriculi and macula
sacculi, through intact round-window permeation was successful for
delivering double stranded siRNAs in vivo for treating various
inner ear ailments and preservation of hearing function. About 40
.mu.l of 10 mM RNA may be contemplated as the dosage for
administration to the ear.
[0747] According to Rejali et al. (Hear Res. 2007 June;
228(1-2):180-7), cochlear implant function can be improved by good
preservation of the spiral ganglion neurons, which are the target
of electrical stimulation by the implant and brain derived
neurotrophic factor (BDNF) has previously been shown to enhance
spiral ganglion survival in experimentally deafened ears. Rejali et
al. tested a modified design of the cochlear implant electrode that
includes a coating of fibroblast cells transduced by a viral vector
with a BDNF gene insert. To accomplish this type of ex vivo gene
transfer, Rejali et al. transduced guinea pig fibroblasts with an
adenovirus with a BDNF gene cassette insert, and determined that
these cells secreted BDNF and then attached BDNF-secreting cells to
the cochlear implant electrode via an agarose gel, and implanted
the electrode in the scala tympani. Rejali et al. determined that
the BDNF expressing electrodes were able to preserve significantly
more spiral ganglion neurons in the basal turns of the cochlea
after 48 days of implantation when compared to control electrodes
and demonstrated the feasibility of combining cochlear implant
therapy with ex vivo gene transfer for enhancing spiral ganglion
neuron survival. Such a system may be applied to the nucleic
acid-targeting system of the present invention for delivery to the
ear.
[0748] Mukherjea et al. (Antioxidants & Redox Signaling, Volume
13, Number 5, 2010) document that knockdown of NOX3 using short
interfering (si) RNA abrogated cisplatin ototoxicity, as evidenced
by protection of OHCs from damage and reduced threshold shifts in
auditory brainstem responses (ABRs). Different doses of siNOX3
(0.3, 0.6, and 0.9 .mu.g) were administered to rats and NOX3
expression was evaluated by real time RT-PCR. The lowest dose of
NOX3 siRNA used (0.3 .mu.g) did not show any inhibition of NOX3
mRNA when compared to transtympanic administration of scrambled
siRNA or untreated cochleae. However, administration of the higher
doses of NOX3 siRNA (0.6 and 0.9 .mu.g) reduced NOX3 expression
compared to control scrambled siRNA. Such a system may be applied
to the CRISPR Cas system of the present invention for transtympanic
administration with a dosage of about 2 mg to about 4 mg of CRISPR
Cas for administration to a human.
[0749] Jung et al. (Molecular Therapy, vol. 21 no. 4, 834-841 April
2013) demonstrate that Hes5 levels in the utricle decreased after
the application of siRNA and that the number of hair cells in these
utricles was significantly larger than following control treatment.
The data suggest that siRNA technology may be useful for inducing
repair and regeneration in the inner ear and that the Notch
signaling pathway is a potentially useful target for specific gene
expression inhibition. Jung et al. injected 8 .mu.g of Hes5 siRNA
in 2 .mu.l volume, prepared by adding sterile normal saline to the
lyophilized siRNA to a vestibular epithelium of the ear. Such a
system may be applied to the nucleic acid-targeting system of the
present invention for administration to the vestibular epithelium
of the ear with a dosage of about 1 to about 30 mg of CRISPR Cas
for administration to a human.
Treating Diseases of the Eye
[0750] The present invention also contemplates delivering the
CRISPR-Cas system to one or both eyes.
[0751] In yet another aspect of the invention, the CRISPR-Cas
system may be used to correct ocular defects that arise from
several genetic mutations further described in Genetic Diseases of
the Eye, Second Edition, edited by Elias I. Traboulsi, Oxford
University Press, 2012.
[0752] For administration to the eye, lentiviral vectors, in
particular equine infectious anemia viruses (EIAV) are particularly
preferred.
[0753] In another embodiment, minimal non-primate lentiviral
vectors based on the equine infectious anemia virus (EIAV) are also
contemplated, especially for ocular gene therapy (see, e.g.,
Balagaan, J Gene Med 2006; 8: 275-285, Published online 21 Nov.
2005 in Wiley InterScience (www.interscience.wiley.com). DOI:
10.1002/jgm.845). The vectors are contemplated to have
cytomegalovirus (CMV) promoter driving expression of the target
gene. Intracameral, subretinal, intraocular and intravitreal
injections are all contemplated (see, e.g., Balagaan, J Gene Med
2006; 8: 275-285, Published online 21 Nov. 2005 in Wiley
InterScience (www.interscience.wiley.com). DOI: 10.1002/jgm.845).
Intraocular injections may be performed with the aid of an
operating microscope. For subretinal and intravitreal injections,
eyes may be prolapsed by gentle digital pressure and fundi
visualised using a contact lens system consisting of a drop of a
coupling medium solution on the cornea covered with a glass
microscope slide coverslip. For subretinal injections, the tip of a
10-mm 34-gauge needle, mounted on a 5-.mu.l Hamilton syringe may be
advanced under direct visualisation through the superior equatorial
sclera tangentially towards the posterior pole until the aperture
of the needle was visible in the subretinal space. Then, 2 .mu.l of
vector suspension may be injected to produce a superior bullous
retinal detachment, thus confirming subretinal vector
administration. This approach creates a self-sealing sclerotomy
allowing the vector suspension to be retained in the subretinal
space until it is absorbed by the RPE, usually within 48 h of the
procedure. This procedure may be repeated in the inferior
hemisphere to produce an inferior retinal detachment. This
technique results in the exposure of approximately 70% of
neurosensory retina and RPE to the vector suspension. For
intravitreal injections, the needle tip may be advanced through the
sclera 1 mm posterior to the corneoscleral limbus and 2 .mu.l of
vector suspension injected into the vitreous cavity. For
intracameral injections, the needle tip may be advanced through a
corneoscleral limbal paracentesis, directed towards the central
cornea, and 2 .mu.l of vector suspension may be injected. For
intracameral injections, the needle tip may be advanced through a
corneoscleral limbal paracentesis, directed towards the central
cornea, and 2 .mu.l of vector suspension may be injected. These
vectors may be injected at titres of either 1.0-1.4.times.10.sup.10
or 1.0-1.4.times.10.sup.9 transducing units (TU)/ml.
[0754] In another embodiment, RetinoStat.RTM., an equine infectious
anemia virus-based lentiviral gene therapy vector that expresses
angiostatic proteins endostain and angiostatin that is delivered
via a subretinal injection for the treatment of the web form of
age-related macular degeneration is also contemplated (see, e.g.,
Binley et al., HUMAN GENE THERAPY 23:980-991 (September 2012)).
Such a vector may be modified for the CRISPR-Cas system of the
present invention. Each eye may be treated with either
RetinoStat.RTM. at a dose of 1.1.times.10.sup.5 transducing units
per eye (TU/eye) in a total volume of 100 .mu.l.
[0755] In an embodiment, mention is made of WO 2015/153780 which
comprehends providing a treatment or prevention of Primary Open
Angle Glaucoma (POAG) by targeting the coding sequence of the MYOC
gene. Some of the target mutations which give rise to POAG include,
but are not limited to, P370 (e.g. P370L); 1477 (e.g., I477N or
I477S); T377 (e.g., TE77R); Q368 (Q368stop)--all in the MYOC gene.
The target mutation also may include a mutational hotspot between
amino acid sequence positions 246-252 in the MYOC gene. In an
embodiment, the target mutation is a mutational hotspot between
amino acid sequence positions, e.g., amino acids 368-380, amino
acids 368-370+377-380, amino acids 364-380, or amino acids 347-380
in the MYOC gene. In an embodiment, the target mutation is a
mutational hotspot between amino acid sequence positions 423-437
(e.g., amino acids 423-426, amino acids 423-427 and amino acids
423-437) in the MYOC gene. In an embodiment, the target mutation is
a mutational hotspot between amino acid sequence positions 477-502
in the MYOC gene (see, e.g., WO 2015/153780).
[0756] In another embodiment, an E1-, partial E3-, E4-deleted
adenoviral vector may be contemplated for delivery to the eye.
Twenty-eight patients with advanced neovascular age related macular
degeneration (AMD) were given a single intravitreous injection of
an E1-, partial E3-, E4-deleted adenoviral vector expressing human
pigment epithelium-derived factor (AdPEDF.ll) (see, e.g.,
Campochiaro et al., Human Gene Therapy 17:167-176 (February 2006)).
Doses ranging from 10.sup.6 to 10.sup.9.5 particle units (PU) were
investigated and there were no serious adverse events related to
AdPEDF.ll and no dose-limiting toxicities (see, e.g., Campochiaro
et al., Human Gene Therapy 17:167-176 (February 2006)). Adenoviral
vector mediated ocular gene transfer appears to be a viable
approach for the treatment of ocular disorders and could be applied
to the CRISPR Cas system.
[0757] In another embodiment, the sd-rxRNA.RTM. system of R.times.i
Pharmaceuticals may be used/and or adapted for delivering CRISPR
Cas to the eye. In this system, a single intravitreal
administration of 3 .mu.g of sd-rxRNA results in sequence-specific
reduction of PPM mRNA levels for 14 days. The sd-rxRNA.RTM. system
may be applied to the nucleic acid-targeting system of the present
invention, contemplating a dose of about 3 to 20 mg of CRISPR
administered to a human.
[0758] Millington-Ward et al. (Molecular Therapy, vol. 19 no. 4,
642-649 April 2011) describes adeno-associated virus (AAV) vectors
to deliver an RNA interference (RNAi)-based rhodopsin suppressor
and a codon-modified rhodopsin replacement gene resistant to
suppression due to nucleotide alterations at degenerate positions
over the RNAi target site. An injection of either
6.0.times.10.sup.8 vp or 1.8.times.10.sup.10 vp AAV were
subretinally injected into the eyes by Millington-Ward et al. The
AAV vectors of Millington-Ward et al. may be applied to the CRISPR
Cas system of the present invention, contemplating a dose of about
2.times.10.sup.11 to about 6.times.10.sup.13 vp administered to a
human.
[0759] Dalkara et al. (Sci Transl Med 5, 189ra76 (2013)) also
relates to in vivo directed evolution to fashion an AAV vector that
delivers wild-type versions of defective genes throughout the
retina after noninjurious injection into the eyes' vitreous humor.
Dalkara describes a 7mer peptide display library and an AAV library
constructed by DNA shuffling of cap genes from AAV1, 2, 4, 5, 6, 8,
and 9. The rcAAV libraries and rAAV vectors expressing GFP under a
CAG or Rho promoter were packaged and deoxyribonuclease-resistant
genomic titers were obtained through quantitative PCR. The
libraries were pooled, and two rounds of evolution were performed,
each consisting of initial library diversification followed by
three in vivo selection steps. In each such step, P30 rho-GFP mice
were intravitreally injected with 2 ml of iodixanol-purified,
phosphate-buffered saline (PBS)-dialyzed library with a genomic
titer of about 1.times.10.sup.12 vg/ml. The AAV vectors of Dalkara
et al. may be applied to the nucleic acid-targeting system of the
present invention, contemplating a dose of about 1.times.10.sup.15
to about 1.times.10.sup.16 vg/ml administered to a human.
[0760] In another embodiment, the rhodopsin gene may be targeted
for the treatment of retinitis pigmentosa (RP), wherein the system
of US Patent Publication No. 20120204282 assigned to Sangamo
BioSciences, Inc. may be modified in accordance of the CRISPR Cas
system of the present invention.
[0761] In another embodiment, the methods of US Patent Publication
No. 20130183282 assigned to Cellectis, which is directed to methods
of cleaving a target sequence from the human rhodopsin gene, may
also be modified to the nucleic acid-targeting system of the
present invention.
[0762] US Patent Publication No. 20130202678 assigned to Academia
Sinica relates to methods for treating retinopathies and
sight-threatening ophthalmologic disorders relating to delivering
of the Puf-A gene (which is expressed in retinal ganglion and
pigmented cells of eye tissues and displays a unique anti-apoptotic
activity) to the sub-retinal or intravitreal space in the eye. In
particular, desirable targets are zgc:193933, prdm1a, spata2,
tex10, rbb4, ddx3, zp2.2, Blimp-1 and HtrA2, all of which may be
targeted by the nucleic acid-targeting system of the present
invention.
[0763] Wu (Cell Stem Cell, 13:659-62, 2013) designed a guide RNA
that led Cas9 to a single base pair mutation that causes cataracts
in mice, where it induced DNA cleavage. Then using either the other
wild-type allele or oligos given to the zygotes repair mechanisms
corrected the sequence of the broken allele and corrected the
cataract-causing genetic defect in mutant mouse.
[0764] US Patent Publication No. 20120159653, describes use of zinc
finger nucleases to genetically modify cells, animals and proteins
associated with macular degeneration (MD). Macular degeneration
(MD) is the primary cause of visual impairment in the elderly, but
is also a hallmark symptom of childhood diseases such as Stargardt
disease, Sorsby fundus, and fatal childhood neurodegenerative
diseases, with an age of onset as young as infancy. Macular
degeneration results in a loss of vision in the center of the
visual field (the macula) because of damage to the retina.
Currently existing animal models do not recapitulate major
hallmarks of the disease as it is observed in humans. The available
animal models comprising mutant genes encoding proteins associated
with MD also produce highly variable phenotypes, making
translations to human disease and therapy development
problematic.
[0765] One aspect of US Patent Publication No. 20120159653 relates
to editing of any chromosomal sequences that encode proteins
associated with MD which may be applied to the nucleic
acid-targeting system of the present invention. The proteins
associated with MD are typically selected based on an experimental
association of the protein associated with MD to an MD disorder.
For example, the production rate or circulating concentration of a
protein associated with MD may be elevated or depressed in a
population having an MD disorder relative to a population lacking
the MD disorder. Differences in protein levels may be assessed
using proteomic techniques including but not limited to Western
blot, immunohistochemical staining, enzyme linked immunosorbent
assay (ELISA), and mass spectrometry. Alternatively, the proteins
associated with MD may be identified by obtaining gene expression
profiles of the genes encoding the proteins using genomic
techniques including but not limited to DNA microarray analysis,
serial analysis of gene expression (SAGE), and quantitative
real-time polymerase chain reaction (Q-PCR).
[0766] By way of non-limiting example, proteins associated with MD
include but are not limited to the following proteins: (ABCA4)
ATP-binding cassette, sub-family A (ABC1), member 4 ACHM1
achromatopsia (rod monochromacy) 1 ApoE Apolipoprotein E (ApoE)
C1QTNF5 (CTRP5) C1q and tumor necrosis factor related protein 5
(C1QTNF5) C2 Complement component 2 (C2) C3 Complement components
(C3) CCL2 Chemokine (C-C motif) Ligand 2 (CCL2) CCR2 Chemokine (C-C
motif) receptor 2 (CCR2) CD36 Cluster of Differentiation 36 CFB
Complement factor B CFH Complement factor CFH H CFHR1 complement
factor H-related 1 CFHR3 complement factor H-related 3 CNGB3 cyclic
nucleotide gated channel beta 3 CP ceruloplasmin (CP) CRP C
reactive protein (CRP) CST3 cystatin C or cystatin 3 (CST3) CTSD
Cathepsin D (CTSD) CX3CR1 chemokine (C-X3-C motif) receptor 1
ELOVL4 Elongation of very long chain fatty acids 4 ERCC6 excision
repair crosscomplementing rodent repair deficiency, complementation
group 6 FBLN5 Fibulin-5 FBLN5 Fibulin 5 FBLN6 Fibulin 6 FSCN2
fascin (FSCN2) HMCN1 Hemicentrin 1 HMCN1 hemicentin 1 HTRA1 HtrA
serine peptidase 1 (HTRA1) HTRA1 HtrA serine peptidase 1 IL-6
Interleukin 6 IL-8 Interleukin 8 LOC387715 Hypothetical protein
PLEKHA1 Pleckstrin homology domain containing family A member 1
(PLEKHA1) PROM1 Prominin 1 (PROM1 or CD133) PRPH2 Peripherin-2 RPGR
retinitis pigmentosa GTPase regulator SERPING1 serpin peptidase
inhibitor, clade G, member 1 (C1-inhibitor) TCOF1 Treacle TIMP3
Metalloproteinase inhibitor 3 (TIMP3) TLR3 Toll-like receptor
3.
[0767] The identity of the protein associated with MD whose
chromosomal sequence is edited can and will vary. In preferred
embodiments, the proteins associated with MD whose chromosomal
sequence is edited may be the ATP-binding cassette, sub-family A
(ABC1) member 4 protein (ABCA4) encoded by the ABCR gene, the
apolipoprotein E protein (APOE) encoded by the APOE gene, the
chemokine (C-C motif) Ligand 2 protein (CCL2) encoded by the CCL2
gene, the chemokine (C-C motif) receptor 2 protein (CCR2) encoded
by the CCR2 gene, the ceruloplasmin protein (CP) encoded by the CP
gene, the cathepsin D protein (CTSD) encoded by the CTSD gene, or
the metalloproteinase inhibitor 3 protein (TIMP3) encoded by the
TIMP3 gene. In an exemplary embodiment, the genetically modified
animal is a rat, and the edited chromosomal sequence encoding the
protein associated with MD may be: (ABCA4) ATPbinding cassette,
NM_000350 sub-family A (ABC1), member 4 APOE Apolipoprotein E
NM_138828 (APOE) CCL2 Chemokine (C-C NM_031530 motif) Ligand 2
(CCL2) CCR2 Chemokine (C-C NM_021866 motif) receptor 2 (CCR2) CP
ceruloplasmin (CP) NM_012532 CTSD Cathepsin D (CTSD) NM_134334
TIMP3 Metalloproteinase NM_012886 inhibitor 3 (TIMP3) The animal or
cell may comprise 1, 2, 3, 4, 5, 6, 7 or more disrupted chromosomal
sequences encoding a protein associated with MD and zero, 1, 2, 3,
4, 5, 6, 7 or more chromosomally integrated sequences encoding the
disrupted protein associated with MD.
[0768] The edited or integrated chromosomal sequence may be
modified to encode an altered protein associated with MD. Several
mutations in MD-related chromosomal sequences have been associated
with MD. Non-limiting examples of mutations in chromosomal
sequences associated with MD include those that may cause MD
including in the ABCR protein, E471K (i.e. glutamate at position
471 is changed to lysine), R1129L (i.e. arginine at position 1129
is changed to leucine), T1428M (i.e. threonine at position 1428 is
changed to methionine), R1517S (i.e. arginine at position 1517 is
changed to serine), I1562T (i.e. isoleucine at position 1562 is
changed to threonine), and G1578R (i.e. glycine at position 1578 is
changed to arginine); in the CCR2 protein, V64I (i.e. valine at
position 192 is changed to isoleucine); in CP protein, G969B (i.e.
glycine at position 969 is changed to asparagine or aspartate); in
TIMP3 protein, S156C (i.e. serine at position 156 is changed to
cysteine), G166C (i.e. glycine at position 166 is changed to
cysteine), G167C (i.e. glycine at position 167 is changed to
cysteine), Y168C (i.e. tyrosine at position 168 is changed to
cysteine), S170C (i.e. serine at position 170 is changed to
cysteine), Y172C (i.e. tyrosine at position 172 is changed to
cysteine) and S181C (i.e. serine at position 181 is changed to
cysteine). Other associations of genetic variants in MD-associated
genes and disease are known in the art.
Treating Circulatory and Muscular Diseases
[0769] The present invention also contemplates delivering the
CRISPR-Cas system described herein, e.g. Cas9 effector protein
systems, to the heart. For the heart, a myocardium tropic
adena-associated virus (AAVM) is preferred, in particular AAVM41
which showed preferential gene transfer in the heart (see, e.g.,
Lin-Yanga et al., PNAS, Mar. 10, 2009, vol. 106, no. 10).
Administration may be systemic or local. A dosage of about
1-10.times.10'' vector genomes are contemplated for systemic
administration. See also, e.g., Eulalio et al. (2012) Nature 492:
376 and Somasuntharam et al. (2013) Biomaterials 34: 7790.
[0770] For example, US Patent Publication No. 20110023139,
describes use of zinc finger nucleases to genetically modify cells,
animals and proteins associated with cardiovascular disease.
Cardiovascular diseases generally include high blood pressure,
heart attacks, heart failure, and stroke and TIA. Any chromosomal
sequence involved in cardiovascular disease or the protein encoded
by any chromosomal sequence involved in cardiovascular disease may
be utilized in the methods described in this disclosure. The
cardiovascular-related proteins are typically selected based on an
experimental association of the cardiovascular-related protein to
the development of cardiovascular disease. For example, the
production rate or circulating concentration of a
cardiovascular-related protein may be elevated or depressed in a
population having a cardiovascular disorder relative to a
population lacking the cardiovascular disorder. Differences in
protein levels may be assessed using proteomic techniques including
but not limited to Western blot, immunohistochemical staining,
enzyme linked immunosorbent assay (ELISA), and mass spectrometry.
Alternatively, the cardiovascular-related proteins may be
identified by obtaining gene expression profiles of the genes
encoding the proteins using genomic techniques including but not
limited to DNA microarray analysis, serial analysis of gene
expression (SAGE), and quantitative real-time polymerase chain
reaction (Q-PCR).
Treating Diseases of the Liver and Kidney
[0771] The present invention also contemplates delivering the
CRISPR-Cas system described herein, e.g. Cas9 effector protein
systems, to the liver and/or kidney. Delivery strategies to induce
cellular uptake of the therapeutic nucleic acid include physical
force or vector systems such as viral-, lipid- or complex-based
delivery, or nanocarriers. From the initial applications with less
possible clinical relevance, when nucleic acids were addressed to
renal cells with hydrodynamic high pressure injection systemically,
a wide range of gene therapeutic viral and non-viral carriers have
been applied already to target posttranscriptional events in
different animal kidney disease models in vivo (Csaba Revesz and
Peter Hamar (2011). Delivery Methods to Target RNAs in the Kidney,
Gene Therapy Applications, Prof. Chunsheng Kang (Ed.), ISBN:
978-953-307-541-9, InTech, Available from:
http://www.intechopen.com/books/gene-therapy-applications/delivery-method-
s-to-target-rnas-inthe-kidney). Delivery methods to the kidney may
include those in Yuan et al. (Am J Physiol Renal Physiol 295:
F605-F617, 2008) investigated whether in vivo delivery of small
interfering RNAs (siRNAs) targeting the 12/15-lipoxygenase
(12/15-LO) pathway of arachidonate acid metabolism can ameliorate
renal injury and diabetic nephropathy (DN) in a
streptozotocininjected mouse model of type 1 diabetes. To achieve
greater in vivo access and siRNA expression in the kidney, Yuan et
al. used double-stranded 12/15-LO siRNA oligonucleotides conjugated
with cholesterol. About 400 .mu.g of siRNA was injected
subcutaneously into mice. The method of Yuang et al. may be applied
to the CRISPR Cas system of the present invention contemplating a
1-2 g subcutaneous injection of CRISPR Cas conjugated with
cholesterol to a human for delivery to the kidneys.
[0772] Molitoris et al. (J Am Soc Nephrol 20: 1754-1764, 2009)
exploited proximal tubule cells (PTCs), as the site of
oligonucleotide reabsorption within the kidney to test the efficacy
of siRNA targeted to p53, a pivotal protein in the apoptotic
pathway, to prevent kidney injury. Naked synthetic siRNA to p53
injected intravenously 4 h after ischemic injury maximally
protected both PTCs and kidney function. Molitoris et al.'s data
indicates that rapid delivery of siRNA to proximal tubule cells
follows intravenous administration. For dose-response analysis,
rats were injected with doses of siP53, 0.33; 1, 3, or 5 mg/kg,
given at the same four time points, resulting in cumulative doses
of 1.32; 4, 12, and 20 mg/kg, respectively. All siRNA doses tested
produced a SCr reducing effect on day one with higher doses being
effective over approximately five days compared with PBS-treated
ischemic control rats. The 12 and 20 mg/kg cumulative doses
provided the best protective effect. The method of Molitoris et al.
may be applied to the nucleic acid-targeting system of the present
invention contemplating 12 and 20 mg/kg cumulative doses to a human
for delivery to the kidneys.
[0773] Thompson et al. (Nucleic Acid Therapeutics, Volume 22,
Number 4, 2012) reports the toxicological and pharmacokinetic
properties of the synthetic, small interfering RNA I5NP following
intravenous administration in rodents and nonhuman primates. I5NP
is designed to act via the RNA interference (RNAi) pathway to
temporarily inhibit expression of the pro-apoptotic protein p53 and
is being developed to protect cells from acute ischemia/reperfusion
injuries such as acute kidney injury that can occur during major
cardiac surgery and delayed graft function that can occur following
renal transplantation. Doses of 800 mg/kg I5NP in rodents, and
1,000 mg/kg I5NP in nonhuman primates, were required to elicit
adverse effects, which in the monkey were isolated to direct
effects on the blood that included a sub-clinical activation of
complement and slightly increased clotting times. In the rat, no
additional adverse effects were observed with a rat analogue of
I5NP, indicating that the effects likely represent class effects of
synthetic RNA duplexes rather than toxicity related to the intended
pharmacologic activity of I5NP. Taken together, these data support
clinical testing of intravenous administration of I5NP for the
preservation of renal function following acute ischemia/reperfusion
injury. The no observed adverse effect level (NOAEL) in the monkey
was 500 mg/kg. No effects on cardiovascular, respiratory, and
neurologic parameters were observed in monkeys following i.v.
administration at dose levels up to 25 mg/kg. Therefore, a similar
dosage may be contemplated for intravenous administration of CRISPR
Cas to the kidneys of a human.
[0774] Shimizu et al. (J Am Soc Nephrol 21: 622-633, 2010)
developed a system to target delivery of siRNAs to glomeruli via
poly(ethylene glycol)-poly(L-lysine)-based vehicles. The
siRNA/nanocarrier complex was approximately 10 to 20 nm in
diameter, a size that would allow it to move across the fenestrated
endothelium to access to the mesangium. After intraperitoneal
injection of fluorescence-labeled siRNA/nanocarrier complexes,
Shimizu et al. detected siRNAs in the blood circulation for a
prolonged time. Repeated intraperitoneal administration of a
mitogen-activated protein kinase 1 (MAPK1) siRNA/nanocarrier
complex suppressed glomerular MAPK1 mRNA and protein expression in
a mouse model of glomerulonephritis. For the investigation of siRNA
accumulation, Cy5-labeled siRNAs complexed with PIC nanocarriers
(0.5 ml, 5 nmol of siRNA content), naked Cy5-labeled siRNAs (0.5
ml, 5 nmol), or Cy5-labeled siRNAs encapsulated in HVJ-E (0.5 ml, 5
nmol of siRNA content) were administrated to BALBc mice. The method
of Shimizu et al. may be applied to the nucleic acid-targeting
system of the present invention contemplating a dose of about of
10-20 .mu.mol CRISPR Cas complexed with nanocarriers in about 1-2
liters to a human for intraperitoneal administration and delivery
to the kidneys.
Treating Epithelial and Lung Diseases
[0775] The present invention also contemplates delivering the
CRISPR-Cas system described herein, e.g. Cas9 systems, to one or
both lungs.
[0776] Although AAV-2-based vectors were originally proposed for
CFTR delivery to CF airways, other serotypes such as AAV-1, AAV-5,
AAV-6, and AAV-9 exhibit improved gene transfer efficiency in a
variety of models of the lung epithelium (see, e.g., Li et al.,
Molecular Therapy, vol. 17 no. 12, 2067-277 December 2009). AAV-1
was demonstrated to be .about.100-fold more efficient than AAV-2
and AAV-5 at transducing human airway epithelial cells in vitro, 5
although AAV-1 transduced murine tracheal airway epithelia in vivo
with an efficiency equal to that of AAV-5. Other studies have shown
that AAV-5 is 50-fold more efficient than AAV-2 at gene delivery to
human airway epithelium (HAE) in vitro and significantly more
efficient in the mouse lung airway epithelium in vivo. AAV-6 has
also been shown to be more efficient than AAV-2 in human airway
epithelial cells in vitro and murine airways in vivo. 8 The more
recent isolate, AAV-9, was shown to display greater gene transfer
efficiency than AAV-5 in murine nasal and alveolar epithelia in
vivo with gene expression detected for over 9 months suggesting AAV
may enable long-term gene expression in vivo, a desirable property
for a CFTR gene delivery vector. Furthermore, it was demonstrated
that AAV-9 could be readministered to the murine lung with no loss
of CFTR expression and minimal immune consequences. CF and non-CF
HAE cultures may be inoculated on the apical surface with 100 .mu.l
of AAV vectors for hours (see, e.g., Li et al., Molecular Therapy,
vol. 17 no. 12, 2067-277 December 2009). The MOI may vary from
1.times.10.sup.3 to 4.times.10.sup.5 vector genomes/cell, depending
on virus concentration and purposes of the experiments. The above
cited vectors are contemplated for the delivery and/or
administration of the invention.
[0777] Zamora et al. (Am J Respir Crit Care Med Vol 183. pp
531-538, 2011) reported an example of the application of an RNA
interference therapeutic to the treatment of human infectious
disease and also a randomized trial of an antiviral drug in
respiratory syncytial virus (RSV)-infected lung transplant
recipients. Zamora et al. performed a randomized, double-blind,
placebo controlled trial in LTX recipients with RSV respiratory
tract infection. Patients were permitted to receive standard of
care for RSV. Aerosolized ALN-RSV01 (0.6 mg/kg) or placebo was
administered daily for 3 days. This study demonstrates that an RNAi
therapeutic targeting RSV can be safely administered to LTX
recipients with RSV infection. Three daily doses of ALN-RSV01 did
not result in any exacerbation of respiratory tract symptoms or
impairment of lung function and did not exhibit any systemic
proinflammatory effects, such as induction of cytokines or CRP.
Pharmacokinetics showed only low, transient systemic exposure after
inhalation, consistent with preclinical animal data showing that
ALN-RSV01, administered intravenously or by inhalation, is rapidly
cleared from the circulation through exonuclease mediated digestion
and renal excretion. The method of Zamora et al. may be applied to
the nucleic acid-targeting system of the present invention and an
aerosolized CRISPR Cas, for example with a dosage of 0.6 mg/kg, may
be contemplated for the present invention.
[0778] Schwank et al. (Cell Stem Cell, 13:653-58, 2013) used
CRISPR-Cas9 to correct a defect associated with cystic fibrosis in
human stem cells. The team's target was the gene for an ion
channel, cystic fibrosis transmembrane conductor receptor (CFTR). A
deletion in CFTR causes the protein to misfold in cystic fibrosis
patients. Using cultured intestinal stem cells developed from cell
samples from two children with cystic fibrosis, Schwank et al. were
able to correct the defect using CRISPR along with a donor plasmid
containing the reparative sequence to be inserted. The researchers
then grew the cells into intestinal "organoids," or miniature guts,
and showed that they functioned normally. In this case, about half
of clonal organoids underwent the proper genetic correction.
Treating Diseases of the Muscular System
[0779] The present invention also contemplates delivering the
CRISPR-Cas system described herein, e.g. Cas9 systems, to
muscle(s).
[0780] Bortolanza et al. (Molecular Therapy vol. 19 no. 11,
2055-264 November 2011) shows that systemic delivery of RNA
interference expression cassettes in the FRG1 mouse, after the
onset of facioscapulohumeral muscular dystrophy (FSHD), led to a
dose-dependent long-term FRG1 knockdown without signs of toxicity.
Bortolanza et al. found that a single intravenous injection of
5.times.10.sup.12 vg of rAAV6-sh1FRG1 rescues muscle histopathology
and muscle function of FRG1 mice. In detail, 200 .mu.l containing
2.times.10.sup.12 or 5.times.10.sup.12 vg of vector in
physiological solution were injected into the tail vein using a
25-gauge Terumo syringe. The method of Bortolanza et al. may be
applied to an AAV expressing CRISPR Cas and injected into humans at
a dosage of about 2.times.10.sup.15 or 2.times.10.sup.16 vg of
vector.
[0781] Dumonceaux et al. (Molecular Therapy vol. 18 no. 5, 881-887
May 2010) inhibit the myostatin pathway using the technique of RNA
interference directed against the myostatin receptor AcvRIIb mRNA
(sh-AcvRIIb). The restoration of a quasi-dystrophin was mediated by
the vectorized U7 exon-skipping technique (U7-DYS).
Adeno-associated vectors carrying either the sh-AcvrIIb construct
alone, the U7-DYS construct alone, or a combination of both
constructs were injected in the tibialis anterior (TA) muscle of
dystrophic mdx mice. The injections were performed with 10.sup.11
AAV viral genomes. The method of Dumonceaux et al. may be applied
to an AAV expressing CRISPR Cas and injected into humans, for
example, at a dosage of about 10.sup.14 to about 10.sup.15 vg of
vector.
[0782] Kinouchi et al. (Gene Therapy (2008) 15, 1126-1130) report
the effectiveness of in vivo siRNA delivery into skeletal muscles
of normal or diseased mice through nanoparticle formation of
chemically unmodified siRNAs with atelocollagen (ATCOL).
ATCOL-mediated local application of siRNA targeting myostatin, a
negative regulator of skeletal muscle growth, in mouse skeletal
muscles or intravenously, caused a marked increase in the muscle
mass within a few weeks after application. These results imply that
ATCOL-mediated application of siRNAs is a powerful tool for future
therapeutic use for diseases including muscular atrophy. MstsiRNAs
(final concentration, 10 mM) were mixed with ATCOL (final
concentration for local administration, 0.5%) (AteloGene, Kohken,
Tokyo, Japan) according to the manufacturer's instructions. After
anesthesia of mice (20-week-old male C57BL/6) by Nembutal (25
mg/kg, i.p.), the Mst-siRNA/ATCOL complex was injected into the
masseter and biceps femoris muscles. The method of Kinouchi et al.
may be applied to CRISPR Cas and injected into a human, for
example, at a dosage of about 500 to 1000 ml of a 40 .mu.M solution
into the muscle. Hagstrom et al. (Molecular Therapy Vol. 10, No. 2,
August 2004) describe an intravascular, nonviral methodology that
enables efficient and repeatable delivery of nucleic acids to
muscle cells (myofibers) throughout the limb muscles of mammals.
The procedure involves the injection of naked plasmid DNA or siRNA
into a distal vein of a limb that is transiently isolated by a
tourniquet or blood pressure cuff. Nucleic acid delivery to
myofibers is facilitated by its rapid injection in sufficient
volume to enable extravasation of the nucleic acid solution into
muscle tissue. High levels of transgene expression in skeletal
muscle were achieved in both small and large animals with minimal
toxicity. Evidence of siRNA delivery to limb muscle was also
obtained. For plasmid DNA intravenous injection into a rhesus
monkey, a threeway stopcock was connected to two syringe pumps
(Model PHD 2000; Harvard Instruments), each loaded with a single
syringe. Five minutes after a papaverine injection, pDNA (15.5 to
25.7 mg in 40-100 ml saline) was injected at a rate of 1.7 or 2.0
ml/s. This could be scaled up for plasmid DNA expressing CRISPR Cas
of the present invention with an injection of about 300 to 500 mg
in 800 to 2000 ml saline for a human. For adenoviral vector
injections into a rat, 2.times.10.sup.9 infectious particles were
injected in 3 ml of normal saline solution (NSS). This could be
scaled up for an adenoviral vector expressing CRISPR Cas of the
present invention with an injection of about 1.times.10.sup.13
infectious particles were injected in 10 liters of NSS for a human.
For siRNA, a rat was injected into the great saphenous vein with
12.5 .mu.g of a siRNA and a primate was injected into the great
saphenous vein with 750 .mu.g of a siRNA. This could be scaled up
for a CRISPR Cas of the present invention, for example, with an
injection of about 15 to about 50 mg into the great saphenous vein
of a human.
[0783] See also, for example, WO2013163628 A2, Genetic Correction
of Mutated Genes, published application of Duke University
describes efforts to correct, for example, a frameshift mutation
which causes a premature stop codon and a truncated gene product
that can be corrected via nuclease mediated non-homologous end
joining such as those responsible for Duchenne Muscular Dystrophy,
("DMD") a recessive, fatal, X-linked disorder that results in
muscle degeneration due to mutations in the dystrophin gene. The
majority of dystrophin mutations that cause DMD are deletions of
exons that disrupt the reading frame and cause premature
translation termination in the dystrophin gene. Dystrophin is a
cytoplasmic protein that provides structural stability to the
dystroglycan complex of the cell membrane that is responsible for
regulating muscle cell integrity and function. The dystrophin gene
or "DMD gene" as used interchangeably herein is 2.2 megabases at
locus Xp21. The primary transcription measures about 2,400 kb with
the mature mRNA being about 14 kb. 79 exons code for the protein
which is over 3500 amino acids. Exon 51 is frequently adjacent to
frame-disrupting deletions in DMD patients and has been targeted in
clinical trials for oligonucleotide-based exon skipping. A clinical
trial for the exon 51 skipping compound eteplirsen recently
reported a significant functional benefit across 48 weeks, with an
average of 47% dystrophin positive fibers compared to baseline.
Mutations in exon 51 are ideally suited for permanent correction by
NHEJ-based genome editing.
[0784] The methods of US Patent Publication No. 20130145487
assigned to Cellectis, which relates to meganuclease variants to
cleave a target sequence from the human dystrophin gene (DMD), may
also be modified to for the nucleic acid-targeting system of the
present invention.
Treating Diseases of the Skin
[0785] The present invention also contemplates delivering the
CRISPR-Cas system described herein, e.g. Cas9 effector protein
systems, to the skin.
[0786] Hickerson et al. (Molecular Therapy--Nucleic Acids (2013) 2,
e129) relates to a motorized microneedle array skin delivery device
for delivering self-delivery (sd)-siRNA to human and murine skin.
The primary challenge to translating siRNA-based skin therapeutics
to the clinic is the development of effective delivery systems.
Substantial effort has been invested in a variety of skin delivery
technologies with limited success. In a clinical study in which
skin was treated with siRNA, the exquisite pain associated with the
hypodermic needle injection precluded enrollment of additional
patients in the trial, highlighting the need for improved, more
"patient-friendly" (i.e., little or no pain) delivery approaches.
Microneedles represent an efficient way to deliver large charged
cargos including siRNAs across the primary barrier, the stratum
corneum, and are generally regarded as less painful than
conventional hypodermic needles. Motorized "stamp type" microneedle
devices, including the motorized microneedle array (MMNA) device
used by Hickerson et al., have been shown to be safe in hairless
mice studies and cause little or no pain as evidenced by (i)
widespread use in the cosmetic industry and (ii) limited testing in
which nearly all volunteers found use of the device to be much less
painful than a flushot, suggesting siRNA delivery using this device
will result in much less pain than was experienced in the previous
clinical trial using hypodermic needle injections. The MMNA device
(marketed as Triple-M or Tri-M by Bomtech Electronic Co, Seoul,
South Korea) was adapted for delivery of siRNA to mouse and human
skin. sd-siRNA solution (up to 300 .mu.l of 0.1 mg/ml RNA) was
introduced into the chamber of the disposable Tri-M needle
cartridge (Bomtech), which was set to a depth of 0.1 mm. For
treating human skin, deidentified skin (obtained immediately
following surgical procedures) was manually stretched and pinned to
a cork platform before treatment. All intradermal injections were
performed using an insulin syringe with a 28-gauge 0.5-inch needle.
The MMNA device and method of Hickerson et al. could be used and/or
adapted to deliver the CRISPR Cas of the present invention, for
example, at a dosage of up to 300 .mu.l of 0.1 mg/ml CRISPR Cas to
the skin.
[0787] Leachman et al. (Molecular Therapy, vol. 18 no. 2, 442-446
February 2010) relates to a phase Ib clinical trial for treatment
of a rare skin disorder pachyonychia congenita (PC), an autosomal
dominant syndrome that includes a disabling plantar keratoderma,
utilizing the first short-interfering RNA (siRNA)-based therapeutic
for skin. This siRNA, called TD101, specifically and potently
targets the keratin 6a (K6a) N171K mutant mRNA without affecting
wild-type K6a mRNA.
[0788] Zheng et al. (PNAS, Jul. 24, 2012, vol. 109, no. 30,
11975-11980) show that spherical nucleic acid nanoparticle
conjugates (SNA-NCs), gold cores surrounded by a dense shell of
highly oriented, covalently immobilized siRNA, freely penetrate
almost 100% of keratinocytes in vitro, mouse skin, and human
epidermis within hours after application. Zheng et al. demonstrated
that a single application of 25 nM epidermal growth factor receptor
(EGFR) SNA-NCs for 60 h demonstrate effective gene knockdown in
human skin. A similar dosage may be contemplated for CRISPR Cas
immobilized in SNA-NCs for administration to the skin.
General Gene Therapy Considerations
[0789] Examples of disease-associated genes and polynucleotides and
disease specific information is available from McKusick-Nathans
Institute of Genetic Medicine, Johns Hopkins University (Baltimore,
Md.) and National Center for Biotechnology Information, National
Library of Medicine (Bethesda, Md.), available on the World Wide
Web.
[0790] Mutations in these genes and pathways can result in
production of improper proteins or proteins in improper amounts
which affect function. Further examples of genes, diseases and
proteins are hereby incorporated by reference from U.S. Provisional
application 61/736,527 filed Dec. 12, 2012. Such genes, proteins
and pathways may be the target polynucleotide of a CRISPR complex
of the present invention.
[0791] Embodiments of the invention also relate to methods and
compositions related to knocking out genes, amplifying genes and
repairing particular mutations associated with DNA repeat
instability and neurological disorders (Robert D. Wells, Tetsuo
Ashizawa, Genetic Instabilities and Neurological Diseases, Second
Edition, Academic Press, Oct. 13, 2011--Medical). Specific aspects
of tandem repeat sequences have been found to be responsible for
more than twenty human diseases (New insights into repeat
instability: role of RNA.cndot.DNA hybrids. Mclvor E I, Polak U,
Napierala M. RNA Biol. 2010 September-October; 7(5):551-8). The
present effector protein systems may be harnessed to correct these
defects of genomic instability.
[0792] Several further aspects of the invention relate to
correcting defects associated with a wide range of genetic diseases
which are further described on the website of the National
Institutes of Health under the topic subsection Genetic Disorders
(website at health.nih.gov/topic/GeneticDisorders). The genetic
brain diseases may include but are not limited to
Adrenoleukodystrophy, Agenesis of the Corpus Callosum, Aicardi
Syndrome, Alpers' Disease, Alzheimer's Disease, Barth Syndrome,
Batten Disease, CADASIL, Cerebellar Degeneration, Fabry's Disease,
Gerstmann-Straussler-Scheinker Disease, Huntington's Disease and
other Triplet Repeat Disorders, Leigh's Disease, Lesch-Nyhan
Syndrome, Menkes Disease, Mitochondrial Myopathies and NINDS
Colpocephaly. These diseases are further described on the website
of the National Institutes of Health under the subsection Genetic
Brain Disorders.
Selected Other Conditions
[0793] Herpes Simplex Virus 1 and 2
[0794] In some embodiments, the treatment, prophylaxis or diagnosis
of HSV-1 (Herpes Simplex Virus 1) is provided. The target is
preferably the UL19, UL30, UL48 or UL50 gene in HSV-1. This is
described in WO2015153789, the disclosure of which is hereby
incorporated by reference.
[0795] In other embodiments, the treatment, prophylaxis or
diagnosis of HSV-2 (Herpes Simplex Virus 2) is provided. The target
is preferably the UL19, UL30, UL48 or UL50 gene in HSV-2. This is
described in WO2015153791, the disclosure of which is hereby
incorporated by reference.
[0796] As the invention involves DD-CRISPR enzymes, DD-Cas,
DD-Cas9, DD-CRISPR-Cas or DD-CRISPR-Cas9 systems or complexes, the
terms "CRISPR", "Cas", "Cas9, "CRISPR system", "CRISPR complex",
"CRISPR-Cas", "CRISPR-Cas9" or the like, without the prefix "DD"
may be considered as having the prefix DD, especially when the
context permits so that the disclosure is reading on DD
embodiments. Thus, in one aspect, the invention provides methods
for using one or more elements of a CRISPR system (which can be
read as DD-CRISPR system and/or CRISPR system"). The CRISPR complex
of the invention provides an effective means for modifying a target
polynucleotide. The CRISPR complex of the invention has a wide
variety of utility including modifying (e.g., deleting, inserting,
translocating, inactivating, activating) a target polynucleotide in
a multiplicity of cell types. As such the CRISPR complex of the
invention has a broad spectrum of applications in, e.g., gene
therapy, drug screening, disease diagnosis, and prognosis.
[0797] With respect to general information on CRISPR-Cas Systems,
components thereof, and delivery of such components, including
methods, materials, delivery vehicles, vectors, particles, AAV, and
making and using thereof, including as to amounts and formulations,
all useful in the practice of the instant invention, reference is
made to: U.S. Pat. Nos. 8,697,359, 8,771,945, 8,795,965, 8,865,406,
8,871,445, 8,889,356, 8,889,418, 8,895,308, 8,906,616, 8,932,814,
8,945,839, 8,993,233 and 8,999,641; US Patent Publications US
2014-0310830 (U.S. application Ser. No. 14/105,031), US
2014-0287938 A1 (U.S. application Ser. No. 14/213,991), US
2014-0273234 A1 (U.S. application Ser. No. 14/293,674),
US2014-0273232 A1 (U.S. application Ser. No. 14/290,575), US
2014-0273231 (U.S. application Ser. No. 14/259,420), US
2014-0256046 A1 (U.S. application Ser. No. 14/226,274), US
2014-0248702 A1 (U.S. application Ser. No. 14/258,458), US
2014-0242700 A1 (U.S. application Ser. No. 14/222,930), US
2014-0242699 A1 (U.S. application Ser. No. 14/183,512), US
2014-0242664 A1 (U.S. application Ser. No. 14/104,990), US
2014-0234972 A1 (U.S. application Ser. No. 14/183,471), US
2014-0227787 A1 (U.S. application Ser. No. 14/256,912), US
2014-0189896 A1 (U.S. application Ser. No. 14/105,035), US
2014-0186958 (U.S. application Ser. No. 14/105,017), US
2014-0186919 A1 (U.S. application Ser. No. 14/104,977), US
2014-0186843 A1 (U.S. application Ser. No. 14/104,900), US
2014-0179770 A1 (U.S. application Ser. No. 14/104,837) and US
2014-0179006 A1 (U.S. application Ser. No. 14/183,486), US
2014-0170753 (U.S. application Ser. No. 14/183,429); US
2015-0184139 (U.S. application Ser. No. 14/324,960); Ser. No.
14/054,414 European Patent Applications EP 2 771 468
(EP13818570.7), EP 2 764 103 (EP13824232.6), and EP 2 784 162
(EP14170383.5); and PCT Patent Publications WO 2014/093661
(PCT/US2013/074743), WO 2014/093694 (PCT/US2013/074790), WO
2014/093595 (PCT/US2013/074611), WO 2014/093718
(PCT/US2013/074825), WO 2014/093709 (PCT/US2013/074812), WO
2014/093622 (PCT/US2013/074667), WO 2014/093635
(PCT/US2013/074691), WO 2014/093655 (PCT/US2013/074736), WO
2014/093712 (PCT/US2013/074819), WO 2014/093701
(PCT/US2013/074800), WO 2014/018423 (PCT/US2013/051418), WO
2014/204723 (PCT/US2014/041790), WO 2014/204724
(PCT/US2014/041800), WO 2014/204725 (PCT/US2014/041803), WO
2014/204726 (PCT/US2014/041804), WO 2014/204727
(PCT/US2014/041806), WO 2014/204728 (PCT/US2014/041808), WO
2014/204729 (PCT/US2014/041809), WO 2015/089351
(PCT/US2014/069897), WO 2015/089354 (PCT/US2014/069902), WO
2015/089364 (PCT/US2014/069925), WO 2015/089427
(PCT/US2014/070068), WO 2015/089462 (PCT/US2014/070127), WO
2015/089419 (PCT/US2014/070057), WO 2015/089465
(PCT/US2014/070135), WO 2015/089486 (PCT/US2014/070175),
PCT/US2015/051691, PCT/US2015/051830. Reference is also made to
U.S. provisional patent applications 61/758,468; 61/802,174;
61/806,375; 61/814,263; 61/819,803 and 61/828,130, filed on Jan.
30, 2013; Mar. 15, 2013; Mar. 28, 2013; Apr. 20, 2013; May 6, 2013
and May 28, 2013 respectively. Reference is also made to U.S.
provisional patent application 61/836,123, filed on Jun. 17, 2013.
Reference is additionally made to U.S. provisional patent
applications 61/835,931, 61/835,936, 61/835,973, 61/836,080,
61/836,101, and 61/836,127, each filed Jun. 17, 2013. Further
reference is made to U.S. provisional patent applications
61/862,468 and 61/862,355 filed on Aug. 5, 2013; 61/871,301 filed
on Aug. 28, 2013; 61/960,777 filed on Sep. 25, 2013 and 61/961,980
filed on Oct. 28, 2013. Reference is yet further made to:
PCT/US2014/62558 filed Oct. 28, 2014, and U.S. Provisional Patent
Application Ser. Nos. 61/915,148, 61/915,150, 61/915,153,
61/915,203, 61/915,251, 61/915,301, 61/915,267, 61/915,260, and
61/915,397, each filed Dec. 12, 2013; 61/757,972 and 61/768,959,
filed on Jan. 29, 2013 and Feb. 25, 2013; 62/010,888 and
62/010,879, both filed Jun. 11, 2014; 62/010,329, 62/010,439 and
62/010,441, each filed Jun. 10, 2014; 61/939,228 and 61/939,242,
each filed Feb. 12, 2014; 61/980,012, filed Apr. 15, 2014;
62/038,358, filed Aug. 17, 2014; 62/055,484, 62/055,460 and
62/055,487, each filed Sep. 25, 2014; and 62/069,243, filed Oct.
27, 2014. Reference is made to PCT application designating, inter
alia, the United States, application No. PCT/US14/41806, filed Jun.
10, 2014. Reference is made to U.S. provisional patent application
61/930,214 filed on Jan. 22, 2014. Reference is made to PCT
application designating, inter alia, the United States, application
No. PCT/US14/41806, filed Jun. 10, 2014.
[0798] Mention is also made of U.S. application 62/180,709, 17 Jun.
15, PROTECTED GUIDE RNAS (PGRNAS); U.S. application 62/091,455,
filed, 12 Dec. 14, PROTECTED GUIDE RNAS (PGRNAS); U.S. application
62/096,708, 24 Dec. 14, PROTECTED GUIDE RNAS (PGRNAS); U.S.
applications 62/091,462, 12 Dec. 14, 62/096,324, 23 Dec. 14,
62/180,681, 17 Jun. 2015, and 62/237,496, 5 Oct. 2015, DEAD GUIDES
FOR CRISPR TRANSCRIPTION FACTORS; U.S. application 62/091,456, 12
Dec. 14 and 62/180,692, 17 Jun. 2015, ESCORTED AND FUNCTIONALIZED
GUIDES FOR CRISPR-CAS SYSTEMS; U.S. application 62/091,461, 12 Dec.
14, DELIVERY, USE AND THERAPEUTIC APPLICATIONS OF THE CRISPR-CAS
SYSTEMS AND COMPOSITIONS FOR GENOME EDITING AS TO HEMATOPOETIC STEM
CELLS (HSCs); U.S. application 62/094,903, 19 Dec. 14, UNBIASED
IDENTIFICATION OF DOUBLE-STRAND BREAKS AND GENOMIC REARRANGEMENT BY
GENOME-WISE INSERT CAPTURE SEQUENCING; U.S. application 62/096,761,
24 Dec. 14, ENGINEERING OF SYSTEMS, METHODS AND OPTIMIZED ENZYME
AND GUIDE SCAFFOLDS FOR SEQUENCE MANIPULATION; U.S. application
62/098,059, 30 Dec. 14, 62/181,641, 18 Jun. 2015, and 62/181,667,
18 Jun. 2015, RNA-TARGETING SYSTEM; U.S. application 62/096,656, 24
Dec. 14 and 62/181,151, 17 Jun. 2015, CRISPR HAVING OR ASSOCIATED
WITH DESTABILIZATION DOMAINS; U.S. application 62/096,697, 24 Dec.
14, CRISPR HAVING OR ASSOCIATED WITH AAV; U.S. application
62/098,158, 30 Dec. 14, ENGINEERED CRISPR COMPLEX INSERTIONAL
TARGETING SYSTEMS; U.S. application 62/151,052, 22 Apr. 15,
CELLULAR TARGETING FOR EXTRACELLULAR EXOSOMAL REPORTING; U.S.
application 62/054,490, 24 Sep. 14, DELIVERY, USE AND THERAPEUTIC
APPLICATIONS OF THE CRISPR-CAS SYSTEMS AND COMPOSITIONS FOR
TARGETING DISORDERS AND DISEASES USING PARTICLE DELIVERY
COMPONENTS; U.S. application 61/939,154, 12 Feb. 14, SYSTEMS,
METHODS AND COMPOSITIONS FOR SEQUENCE MANIPULATION WITH OPTIMIZED
FUNCTIONAL CRISPR-CAS SYSTEMS; U.S. application 62/055,484, 25 Sep.
14, SYSTEMS, METHODS AND COMPOSITIONS FOR SEQUENCE MANIPULATION
WITH OPTIMIZED FUNCTIONAL CRISPR-CAS SYSTEMS; U.S. application
62/087,537, 4 Dec. 14, SYSTEMS, METHODS AND COMPOSITIONS FOR
SEQUENCE MANIPULATION WITH OPTIMIZED FUNCTIONAL CRISPR-CAS SYSTEMS;
U.S. application 62/054,651, 24 Sep. 14, DELIVERY, USE AND
THERAPEUTIC APPLICATIONS OF THE CRISPR-CAS SYSTEMS AND COMPOSITIONS
FOR MODELING COMPETITION OF MULTIPLE CANCER MUTATIONS IN VIVO; U.S.
application 62/067,886, 23 Oct. 14, DELIVERY, USE AND THERAPEUTIC
APPLICATIONS OF THE CRISPR-CAS SYSTEMS AND COMPOSITIONS FOR
MODELING COMPETITION OF MULTIPLE CANCER MUTATIONS IN VIVO; U.S.
applications 62/054,675, 24 Sep. 14 and 62/181,002, 17 Jun. 2015,
DELIVERY, USE AND THERAPEUTIC APPLICATIONS OF THE CRISPR-CAS
SYSTEMS AND COMPOSITIONS IN NEURONAL CELLS/TISSUES; U.S.
application 62/054,528, 24 Sep. 14, DELIVERY, USE AND THERAPEUTIC
APPLICATIONS OF THE CRISPR-CAS SYSTEMS AND COMPOSITIONS IN IMMUNE
DISEASES OR DISORDERS; U.S. application 62/055,454, 25 Sep. 14,
DELIVERY, USE AND THERAPEUTIC APPLICATIONS OF THE CRISPR-CAS
SYSTEMS AND COMPOSITIONS FOR TARGETING DISORDERS AND DISEASES USING
CELL PENETRATION PEPTIDES (CPP); U.S. application 62/055,460, 25
Sep. 14, MULTIFUNCTIONAL-CRISPR COMPLEXES AND/OR OPTIMIZED ENZYME
LINKED FUNCTIONAL-CRISPR COMPLEXES; U.S. application 62/087,475, 4
Dec. 14 and 62/181,690, 18 Jun. 2015, FUNCTIONAL SCREENING WITH
OPTIMIZED FUNCTIONAL CRISPR-CAS SYSTEMS; U.S. application
62/055,487, 25 Sep. 14, FUNCTIONAL SCREENING WITH OPTIMIZED
FUNCTIONAL CRISPR-CAS SYSTEMS; U.S. application 62/087,546, 4 Dec.
14 and 62/181,687, 18 Jun. 2015, MULTIFUNCTIONAL CRISPR COMPLEXES
AND/OR OPTIMIZED ENZYME LINKED FUNCTIONAL-CRISPR COMPLEXES; and
U.S. application 62/098,285, 30 Dec. 14, CRISPR MEDIATED IN VIVO
MODELING AND GENETIC SCREENING OF TUMOR GROWTH AND METASTASIS.
[0799] Mention is made of U.S. applications 62/181,659, 18 Jun.
2015 and 62/207,318, 19 Aug. 2015, ENGINEERING AND OPTIMIZATION OF
SYSTEMS, METHODS, ENZYME AND GUIDE SCAFFOLDS OF CAS9 ORTHOLOGS AND
VARIANTS FOR SEQUENCE MANIPULATION. Mention is made of U.S.
applications 62/181,663, 18 Jun. 2015 and 62/245,264, 22 Oct. 2015,
NOVEL CRISPR ENZYMES AND SYSTEMS, U.S. applications 62/181,675, 18
Jun. 2015, and Attorney Docket No. 46783.01.2128, filed 22 Oct.
2015, NOVEL CRISPR ENZYMES AND SYSTEMS, U.S. application
62/232,067, 24 Sep. 2015, U.S. application 62/205,733, 16 Aug.
2015, U.S. application 62/201,542, 5 Aug. 2015, U.S. application
62/193,507, 16 Jul. 2015, and U.S. application 62/181,739, 18 Jun.
2015, each entitled NOVEL CRISPR ENZYMES AND SYSTEMS and of U.S.
application 62/245,270, 22 Oct. 2015, NOVEL CRISPR ENZYMES AND
SYSTEMS. Mention is also made of U.S. application 61/939,256, 12
Feb. 2014, and WO 2015/089473 (PCT/US2014/070152), 12 Dec. 2014,
each entitled ENGINEERING OF SYSTEMS, METHODS AND OPTIMIZED GUIDE
COMPOSITIONS WITH NEW ARCHITECTURES FOR SEQUENCE MANIPULATION.
Mention is also made of PCT/US2015/045504, 15 Aug. 2015, U.S.
application 62/180,699, 17 Jun. 2015, and U.S. application
62/038,358, 17 Aug. 2014, each entitled GENOME EDITING USING CAS9
NICKASES.
[0800] Each of these patents, patent publications, and
applications, and all documents cited therein or during their
prosecution ("appln cited documents") and all documents cited or
referenced in the appln cited documents, together with any
instructions, descriptions, product specifications, and product
sheets for any products mentioned therein or in any document
therein and incorporated by reference herein, are hereby
incorporated herein by reference, and may be employed in the
practice of the invention. All documents (e.g., these patents,
patent publications and applications and the appln cited documents)
are incorporated herein by reference to the same extent as if each
individual document was specifically and individually indicated to
be incorporated by reference. Also with respect to general
information on CRISPR-Cas Systems, mention is made of the following
(also hereby incorporated herein by reference): [0801] Multiplex
genome engineering using CRISPR/Cas systems. Cong, L., Ran, F. A.,
Cox, D., Lin, S., Barretto, R., Habib, N., Hsu, P. D., Wu, X.,
Jiang, W., Marraffini, L. A., & Zhang, F. Science February 15;
339(6121):819-23 (2013); [0802] RNA guided editing of bacterial
genomes using CRISPR-Cas systems. Jiang W., Bikard D., Cox D.,
Zhang F, Marraffini L A. Nat Biotechnol March; 31(3):233-9 (2013);
[0803] One-Step Generation of Mice Carrying Mutations in Multiple
Genes by CRISPR/Cas-Mediated Genome Engineering. Wang H., Yang H.,
Shivalila C S., Dawlaty M M., Cheng A W., Zhang F., Jaenisch R.
Cell May 9; 153(4):910-8 (2013); [0804] Optical control of
mammalian endogenous transcription and epigenetic states. Konermann
S, Brigham M D, Trevino A E, Hsu P D, Heidenreich M, Cong L, Platt
R J, Scott D A, Church G M, Zhang F. Nature. 2013 Aug. 22;
500(7463):472-6. doi: 10.1038/Nature12466. Epub 2013 Aug. 23;
[0805] Double Nicking by RNA-Guided CRISPR Cas9 for Enhanced Genome
Editing Specificity. Ran, F A., Hsu, P D., Lin, C Y., Gootenberg, J
S., Konermann, S., Trevino, A E., Scott, D A., Inoue, A., Matoba,
S., Zhang, Y., & Zhang, F. Cell August 28. pii:
S0092-8674(13)01015-5. (2013); [0806] DNA targeting specificity of
RNA-guided Cas9 nucleases. Hsu, P., Scott, D., Weinstein, J., Ran,
F A., Konermann, S., Agarwala, V., Li, Y., Fine, E., Wu, X.,
Shalem, O., Cradick, T J., Marraffini, L A., Bao, G., & Zhang,
F. Nat Biotechnol doi:10.1038/nbt.2647 (2013); [0807] Genome
engineering using the CRISPR-Cas9 system. Ran, F A., Hsu, P D.,
Wright, J., Agarwala, V., Scott, D A., Zhang, F. Nature Protocols
November; 8(11):2281-308. (2013); [0808] Genome-Scale CRISPR-Cas9
Knockout Screening in Human Cells. Shalem, O., Sanjana, N E.,
Hartenian, E., Shi, X., Scott, D A., Mikkelson, T., Heckl, D.,
Ebert, BL., Root, D E., Doench, J G., Zhang, F. Science December
12. (2013). [Epub ahead of print]; [0809] Crystal structure of cas9
in complex with guide RNA and target DNA. Nishimasu, H., Ran, F A.,
Hsu, P D., Konermann, S., Shehata, S I., Dohmae, N., Ishitani, R.,
Zhang, F., Nureki, O. Cell February 27. (2014). 156(5):935-49;
[0810] Genome-wide binding of the CRISPR endonuclease Cas9 in
mammalian cells. Wu X., Scott D A., Kriz A J., Chiu A C., Hsu P D.,
Dadon D B., Cheng A W., Trevino A E., Konermann S., Chen S.,
Jaenisch R., Zhang F., Sharp P A. Nat Biotechnol. (2014) April 20.
doi: 10.1038/nbt.2889, [0811] CRISPR-Cas9 Knockin Mice for Genome
Editing and Cancer Modeling, Platt et al., Cell 159(2): 440-455
(2014) DOI: 10.1016/j.cell.2014.09.014, [0812] Development and
Applications of CRISPR-Cas9 for Genome Engineering, Hsu et al, Cell
157, 1262-1278 (Jun. 5, 2014) (Hsu 2014), [0813] Genetic screens in
human cells using the CRISPR/Cas9 system, Wang et al., Science.
2014 Jan. 3; 343(6166): 80-84. doi:10.1126/science.1246981, [0814]
Rational design of highly active sgRNAs for CRISPR-Cas9-mediated
gene inactivation, Doench et al., Nature Biotechnology published
online 3 Sep. 2014; doi:10.1038/nbt.3026, and [0815] In vivo
interrogation of gene function in the mammalian brain using
CRISPR-Cas9, Swiech et al, Nature Biotechnology; published online
19 Oct. 2014; doi:10.1038/nbt.3055. [0816] Genome-scale
transcriptional activation by an engineered CRISPR-Cas9 complex,
Konermann S, Brigham M D, Trevino A E, Joung J, Abudayyeh O O,
Barcena C, Hsu P D, Habib N, Gootenberg J S, Nishimasu H, Nureki O,
Zhang F., Nature. January 29; 517(7536):583-8 (2015). [0817] A
split-Cas9 architecture for inducible genome editing and
transcription modulation, Zetsche B, Volz S E, Zhang F., (published
online 2 Feb. 2015) Nat Biotechnol. February; 33(2):139-42 (2015);
[0818] Genome-wide CRISPR Screen in a Mouse Model of Tumor Growth
and Metastasis, Chen S, Sanjana N E, Zheng K, Shalem O, Lee K, Shi
X, Scott D A, Song J, Pan J Q, Weissleder R, Lee H, Zhang F, Sharp
P A. Cell 160, 1246-1260, Mar. 12, 2015 (multiplex screen in
mouse), and [0819] In vivo genome editing using Staphylococcus
aureus Cas9, Ran F A, Cong L, Yan W X, Scott D A, Gootenberg J S,
Kriz A J, Zetsche B, Shalem O, Wu X, Makarova K S, Koonin E V,
Sharp P A, Zhang F., (published online 1 Apr. 2015), Nature. April
9; 520(7546):186-91 (2015). [0820] High-throughput functional
genomics using CRISPR-Cas9, Shalem et al., Nature Reviews Genetics
16, 299-311 (May 2015). [0821] Sequence determinants of improved
CRISPR sgRNA design, Xu et al., Genome Research 25, 1147-1157
(August 2015). [0822] A Genome-wide CRISPR Screen in Primary Immune
Cells to Dissect Regulatory Networks, Parnas et al., Cell 162,
675-686 (Jul. 30, 2015). [0823] CRISPR/Cas9 cleavage of viral DNA
efficiently suppresses hepatitis B virus, Ramanan et al.,
Scientific Reports 5:10833. doi: 10.1038/srep10833 (Jun. 2, 2015).
[0824] Crystal Structure of Staphylococcus aureus Cas9, Nishimasu
et al., Cell 162, 1113-1126 (Aug. 27, 2015). [0825] 71.BCL11A
enhancer dissection by Cas9-mediated in situ saturating
mutagenesis, Canver et al., Nature 527(7577):192-7 (Nov. 12, 2015)
doi: 10.1038/nature15521. Epub 2015 Sep. 16. [0826] Cpf1 Is a
Single RNA-Guided Endonuclease of a Class 2 CRISPR-Cas System,
Zetsche et al., Cell 163, 759-71 (Sep. 25, 2015). [0827] Discovery
and Functional Characterization of Diverse Class 2 CRISPR-Cas
Systems, Shmakov et al., Molecular Cell, 60(3), 385-397 doi:
10.1016/j.molcel.2015.10.008 Epub Oct. 22, 2015. each of which is
incorporated herein by reference, and discussed briefly below:
[0828] Cong et al. engineered type II CRISPR/Cas systems for use in
eukaryotic cells based on both Streptococcus thermophilus Cas9 and
also Streptococcus pyogenes Cas9 and demonstrated that Cas9
nucleases can be directed by short RNAs to induce precise cleavage
of DNA in human and mouse cells. Their study further showed that
Cas9 as converted into a nicking enzyme can be used to facilitate
homology-directed repair in eukaryotic cells with minimal mutagenic
activity. Additionally, their study demonstrated that multiple
guide sequences can be encoded into a single CRISPR array to enable
simultaneous editing of several at endogenous genomic loci sites
within the mammalian genome, demonstrating easy programmability and
wide applicability of the RNA-guided nuclease technology. This
ability to use RNA to program sequence specific DNA cleavage in
cells defined a new class of genome engineering tools. These
studies further showed that other CRISPR loci are likely to be
transplantable into mammalian cells and can also mediate mammalian
genome cleavage. Importantly, it can be envisaged that several
aspects of the CRISPR/Cas system can be further improved to
increase its efficiency and versatility. [0829] Jiang et al. used
the clustered, regularly interspaced, short palindromic repeats
(CRISPR)-associated Cas9 endonuclease complexed with dual-RNAs to
introduce precise mutations in the genomes of Streptococcus
pneumoniae and Escherichia coli. The approach relied on
dual-RNA:Cas9-directed cleavage at the targeted genomic site to
kill unmutated cells and circumvents the need for selectable
markers or counter-selection systems. The study reported
reprogramming dual-RNA:Cas9 specificity by changing the sequence of
short CRISPR RNA (crRNA) to make single- and multinucleotide
changes carried on editing templates. The study showed that
simultaneous use of two crRNAs enabled multiplex mutagenesis.
Furthermore, when the approach was used in combination with
recombineering, in S. pneumoniae, nearly 100% of cells that were
recovered using the described approach contained the desired
mutation, and in E. coli, 65% that were recovered contained the
mutation. [0830] Wang et al. (2013) used the CRISPR/Cas system for
the one-step generation of mice carrying mutations in multiple
genes which were traditionally generated in multiple steps by
sequential recombination in embryonic stem cells and/or
time-consuming intercrossing of mice with a single mutation. The
CRISPR/Cas system will greatly accelerate the in vivo study of
functionally redundant genes and of epistatic gene interactions.
[0831] Konermann et al. addressed the need in the art for versatile
and robust technologies that enable optical and chemical modulation
of DNA-binding domains based CRISPR Cas9 enzyme and also
Transcriptional Activator Like Effectors. [0832] Ran et al.
(2013-A) described an approach that combined a Cas9 nickase mutant
with paired guide RNAs to introduce targeted double-strand breaks.
This addresses the issue of the Cas9 nuclease from the microbial
CRISPR-Cas system being targeted to specific genomic loci by a
guide sequence, which can tolerate certain mismatches to the DNA
target and thereby promote undesired off-target mutagenesis.
Because individual nicks in the genome are repaired with high
fidelity, simultaneous nicking via appropriately offset guide RNAs
is required for double-stranded breaks and extends the number of
specifically recognized bases for target cleavage. The authors
demonstrated that using paired nicking can reduce off-target
activity by 50- to 1,500-fold in cell lines and to facilitate gene
knockout in mouse zygotes without sacrificing on-target cleavage
efficiency. This versatile strategy enables a wide variety of
genome editing applications that require high specificity. [0833]
Hsu et al. (2013) characterized SpCas9 targeting specificity in
human cells to inform the selection of target sites and avoid
off-target effects. The study evaluated >700 guide RNA variants
and SpCas9-induced indel mutation levels at >100 predicted
genomic off-target loci in 293T and 293FT cells. The authors that
SpCas9 tolerates mismatches between guide RNA and target DNA at
different positions in a sequence-dependent manner, sensitive to
the number, position and distribution of mismatches. The authors
further showed that SpCas9-mediated cleavage is unaffected by DNA
methylation and that the dosage of SpCas9 and sgRNA can be titrated
to minimize off-target modification. Additionally, to facilitate
mammalian genome engineering applications, the authors reported
providing a web-based software tool to guide the selection and
validation of target sequences as well as off-target analyses.
[0834] Ran et al. (2013-B) described a set of tools for
Cas9-mediated genome editing via non-homologous end joining (NHEJ)
or homology-directed repair (HDR) in mammalian cells, as well as
generation of modified cell lines for downstream functional
studies. To minimize off-target cleavage, the authors further
described a double-nicking strategy using the Cas9 nickase mutant
with paired guide RNAs. The protocol provided by the authors
experimentally derived guidelines for the selection of target
sites, evaluation of cleavage efficiency and analysis of off-target
activity. The studies showed that beginning with target design,
gene modifications can be achieved within as little as 1-2 weeks,
and modified clonal cell lines can be derived within 2-3 weeks.
[0835] Shalem et al. described a new way to interrogate gene
function on a genome-wide scale. Their studies showed that delivery
of a genome-scale CRISPR-Cas9 knockout (GeCKO) library targeted
18,080 genes with 64,751 unique guide sequences enabled both
negative and positive selection screening in human cells. First,
the authors showed use of the GeCKO library to identify genes
essential for cell viability in cancer and pluripotent stem cells.
Next, in a melanoma model, the authors screened for genes whose
loss is involved in resistance to vemurafenib, a therapeutic that
inhibits mutant protein kinase BRAF. Their studies showed that the
highest-ranking candidates included previously validated genes NF1
and MED12 as well as novel hits NF2, CUL3, TADA2B, and TADA1. The
authors observed a high level of consistency between independent
guide RNAs targeting the same gene and a high rate of hit
confirmation, and thus demonstrated the promise of genome-scale
screening with Cas9. [0836] Nishimasu et al. reported the crystal
structure of Streptococcus pyogenes Cas9 in complex with sgRNA and
its target DNA at 2.5 A.degree. resolution. The structure revealed
a bilobed architecture composed of target recognition and nuclease
lobes, accommodating the sgRNA:DNA heteroduplex in a positively
charged groove at their interface. Whereas the recognition lobe is
essential for binding sgRNA and DNA, the nuclease lobe contains the
HNH and RuvC nuclease domains, which are properly positioned for
cleavage of the complementary and non-complementary strands of the
target DNA, respectively. The nuclease lobe also contains a
carboxyl-terminal domain responsible for the interaction with the
protospacer adjacent motif (PAM). This high-resolution structure
and accompanying functional analyses have revealed the molecular
mechanism of RNA-guided DNA targeting by Cas9, thus paving the way
for the rational design of new, versatile genome-editing
technologies. [0837] Wu et al. mapped genome-wide binding sites of
a catalytically inactive Cas9 (dCas9) from Streptococcus pyogenes
loaded with single guide RNAs (sgRNAs) in mouse embryonic stem
cells (mESCs). The authors showed that each of the four sgRNAs
tested targets dCas9 to between tens and thousands of genomic
sites, frequently characterized by a 5-nucleotide seed region in
the sgRNA and an NGG protospacer adjacent motif (PAM). Chromatin
inaccessibility decreases dCas9 binding to other sites with
matching seed sequences; thus 70% of off-target sites are
associated with genes. The authors showed that targeted sequencing
of 295 dCas9 binding sites in mESCs transfected with catalytically
active Cas9 identified only one site mutated above background
levels. The authors proposed a two-state model for Cas9 binding and
cleavage, in which a seed match triggers binding but extensive
pairing with target DNA is required for cleavage. [0838] Platt et
al. established a Cre-dependent Cas9 knockin mouse. The authors
demonstrated in vivo as well as ex vivo genome editing using
adeno-associated virus (AAV)-, lentivirus-, or particle-mediated
delivery of guide RNA in neurons, immune cells, and endothelial
cells.
[0839] Hsu et al. (2014) is a review article that discusses
generally CRISPR-Cas9 history from yogurt to genome editing,
including genetic screening of cells. [0840] Wang et al. (2014)
relates to a pooled, loss-of-function genetic screening approach
suitable for both positive and negative selection that uses a
genome-scale lentiviral single guide RNA (sgRNA) library. [0841]
Doench et al. created a pool of sgRNAs, tiling across all possible
target sites of a panel of six endogenous mouse and three
endogenous human genes and quantitatively assessed their ability to
produce null alleles of their target gene by antibody staining and
flow cytometry. The authors showed that optimization of the PAM
improved activity and also provided an on-line tool for designing
sgRNAs. [0842] Swiech et al. demonstrate that AAV-mediated SpCas9
genome editing can enable reverse genetic studies of gene function
in the brain. [0843] Konermann et al. (2015) discusses the ability
to attach multiple effector domains, e.g., transcriptional
activator, functional and epigenomic regulators at appropriate
positions on the guide such as stem or tetraloop with and without
linkers. [0844] Zetsche et al. demonstrates that the Cas9 enzyme
can be split into two and hence the assembly of Cas9 for activation
can be controlled. [0845] Chen et al. relates to multiplex
screening by demonstrating that a genome-wide in vivo CRISPR-Cas9
screen in mice reveals genes regulating lung metastasis. [0846] Ran
et al. (2015) relates to SaCas9 and its ability to edit genomes and
demonstrates that one cannot extrapolate from biochemical assays.
Shalem et al. (2015) described ways in which catalytically inactive
Cas9 (dCas9) fusions are used to synthetically repress (CRISPRi) or
activate (CRISPRa) expression, showing. advances using Cas9 for
genome-scale screens, including arrayed and pooled screens,
knockout approaches that inactivate genomic loci and strategies
that modulate transcriptional activity. [0847] Shalem et al. (2015)
described ways in which catalytically inactive Cas9 (dCas9) fusions
are used to synthetically repress (CRISPRi) or activate (CRISPRa)
expression, showing. advances using Cas9 for genome-scale screens,
including arrayed and pooled screens, knockout approaches that
inactivate genomic loci and strategies that modulate
transcriptional activity. [0848] Xu et al. (2015) assessed the DNA
sequence features that contribute to single guide RNA (sgRNA)
efficiency in CRISPR-based screens. The authors explored efficiency
of CRISPR/Cas9 knockout and nucleotide preference at the cleavage
site. The authors also found that the sequence preference for
CRISPRi/a is substantially different from that for CRISPR/Cas9
knockout. [0849] Parnas et al. (2015) introduced genome-wide pooled
CRISPR-Cas9 libraries into dendritic cells (DCs) to identify genes
that control the induction of tumor necrosis factor (Tnf) by
bacterial lipopolysaccharide (LPS). Known regulators of Tlr4
signaling and previously unknown candidates were identified and
classified into three functional modules with distinct effects on
the canonical responses to LPS. [0850] Ramanan et al (2015)
demonstrated cleavage of viral episomal DNA (cccDNA) in infected
cells. The HBV genome exists in the nuclei of infected hepatocytes
as a 3.2 kb double-stranded episomal DNA species called covalently
closed circular DNA (cccDNA), which is a key component in the HBV
life cycle whose replication is not inhibited by current therapies.
The authors showed that sgRNAs specifically targeting highly
conserved regions of HBV robustly suppresses viral replication and
depleted cccDNA. [0851] Nishimasu et al. (2015) reported the
crystal structures of SaCas9 in complex with a single guide RNA
(sgRNA) and its double-stranded DNA targets, containing the
5'-TTGAAT-3' PAM and the 5'-TTGGGT-3' PAM. A structural comparison
of SaCas9 with SpCas9 highlighted both structural conservation and
divergence, explaining their distinct PAM specificities and
orthologous sgRNA recognition. Mention is also made of Tsai et al,
"Dimeric CRISPR RNA-guided Fok1 nucleases for highly specific
genome editing," Nature Biotechnology 32(6): 569-77 (2014) which is
not believed to be prior art to the instant invention or
application, but which may be considered in the practice of the
instant invention. Mention is also made of Konermann et al.,
"Genome-scale transcription activation by an engineered CRISPR-Cas9
complex," doi:10.1038/nature14136, incorporated herein by
reference.
[0852] The present invention will be further illustrated in the
following Examples which are given for illustration purposes only
and are not intended to limit the invention in any way.
EXAMPLES
Example 1: Construction and Testing of DD-Cas9
[0853] Materials and Methods for destabilizing domain Cas9 fusions:
Vector construction: ER50-Cas9, Cas9-ER50, ER50-Cas9-ER50,
DHFR-Cas9, Cas9-DHFR, DHFR-Cas9-DHFR vectors were constructed.
TABLE-US-00013 Sequences of destabilizing domains Amino acid
sequence of N-term DHFR
ISLIAALAVDYVIGMENAMPWNLPADLAWFKRNTLNKPVIMGRHTWESI
GRPLPGRKNIILSSQPSTDDRVTWVKSVDEAIAACGDVPEIMVIGGGRV
IEQFLPKAQKLYLTHIDAEVEGDTHFPDYEPDDWESVFSEFHDADAQNS HSYCFEILERR (SEQ
ID NO: 50) Amino acid sequence of C-term DHFR (sequence difference
from N-term in underlined and in bold)
ISLIAALAVDHVIGMETVMPWNLPADLAWFKRNTLNKPVIMGRHTWESI
GRPLPGRKNIILSSQPSTDDRVTWVKSVDEAIAACGDVPEIMVIGGGRV
YEQFLPKAQKLYLTHIDAEVEGDTHFPDYEPDDWESVFSEFHDADAQNS HSYCFEILERR (SEQ
ID NO: 51) Amino acid sequence of N- and C-term ER50 SLAL
SLTADQMVSALLDAEPPILYSEYDPTRPFSEASMMGLLTNLADR
ELVHMINWAKRVPGFVDLALHDQVHLLECAWMEILMIGLVWRSMEHPGK
LLFAPNLLLDRNQGKCVEGGVEIFDMLLATSSRFRMMNLQGEEFVCLKS
IILLNSGVYTFLSSTLKSLEEKDHIHRVLDKITDTLIHLMAKAGLTLQQ
QHQRLAQLLLILSHIRHMSSKRMEHLYSMKCKNVVPLSDLLLEMLDAHR L (SEQ ID NO:
52)
[0854] Whole Sequence for Constructs
[0855] The amino acid sequence provided here is from Streptococcus
pyogenes (SpCas9). This sequence can be replaced with the sequence
from other Cas9s e.g. SaCas9, StCas9 etc.
TABLE-US-00014 Sequence for ER50-Cas9 Color code for sequence
elements: 3xFlag(MDYKDHDGDYKDHDIDYKDDDDK (SEQ ID NO: 53)) shown in
bold NLS from SV40 (MAPKKKRKVGIHGVPAA (SEQ ID NO: 54)), underlined
Glycine Serine linker (GS and GSGSGS (SEQ ID NO: 55)) shown in bold
in two parts either side of ER50 ER50
(SLALSLTADQMVSALLDAEPPILYSEYDPTRPFSEASMMGLLTNLADRELVHMINWAK
RVPGFVDLALHDQVHLLECAWMEILMIGLVWRSMEHPGKLLFAPNLLLDRNQGKCVE
GGVEIFDMLLATSSRFRMMNLQGEEFVCLKSIILLNSGVYTFLSSTLKSLEEKDHIHRVLD
KITDTLIHLMAKAGLTLQQQHQRLAQLLLILSHIRHMSSKRMEHLYSMKCKNVVPLSDL
LLEMLDAHRL (SEQ ID NO: 52)) SpCas9 (underlined) NLS from
nucleoplasmin (KRPAATKKAGQAKKKK (SEQ ID NO: 2))
MDYKDHDGDYKDHDIDVKDDDDKMAPKKKRKVGIHGVPAAGSSLALSLTADQMVS
ALLDAEPPILYSEYDPTRPFSEASMMGLLTNLADRELVHMINWAKRVPGFVDLALHDQ
VHLLECAWMEILMIGLVWRSMEHPGKLLFAPNLLLDRNQGKCVEGGVEIFDMLLATSS
RFRMMNLQGEEFVCLKSIILLNSGVYTFLSSTLKSLEEKDHIHRVLDKITDTLIHLMAKA
GLTLQQQHQRLAQLLLILSHIRHMSSKRMEHLYSMKCKNVVPLSDLLLEMLDAHRLGS
GSGSDKKYSIGLDIGTNSVGWAVITDEYKVPSKKFKVLGNTDRHSIKKNLIGALLFDSGE
TAEATRLKRTARRRYTRRKNRICYLQEIFSNEMAKVDDSFFHRLEESFLVEEDKKHERH
PIFGNIVDEVAYHEKYPTTYHLRKKLVDSTDKADLRLIYLALAHMIKFRGHFLIEGDLNG
DNSDVDKLFIQLVQTYNQLFEENPINASGVDAKAILSARLSKSRRLENLIAQLPGEKKNG
LFGNLIALSLGLTPNFKSNFDLAEDAKLQLSKDTYDDDLDNLLAQIGDQYADLFLAAKN
LSDAILLSDILRVNTEITKAPLSASMIKRYDEHHQDLTLLKALVRQQLPEKYKEIFFDQSK
NGYAGYIDGGASQEEFYKFIKPILEKMDGTEELLVKLNREDLLRKQRTFDNGSIPHQIHL
GELHAILRRQEDFYPFLKDNREKIEKILTFRIPYYVGPLARGNSRFAWMTRKSEETITPW
NFEEVVDKGASAQSFIERMTNFDKNLPNEKVLPKHSLLYEYFTVYNELTKVKYVTEGM
RKPAFLSGEQKKAIVDLLFKTNRKVTVKQLKEDYFKKIECFDSVEISGVEDRFNASLGTY
HDLLKIIKDKDFLDNEENEDILEDIVLTLTLFEDREMIEERLKTYAHLFDDKVMKQLKRR
RYTGWGRLSRKLINGIRDKQSGKTILDFLKSDGFANRNFMQLIHDDSLTFKEDIQKAQVS
GQGDSLHEHIANLAGSPAIKKGILQTVKVVDELVKVMGRHKPENIVIEMARENQTTQKG
QKNSRERMKRIEEGIKELGSQILKEHPVENTQLQNEKLYLYYLQNGRDMYVDQELDINR
LSDYDVDHIVPQSFLKDDSIDNKVLTRSDKNRGKSDNVPSEEVVKKMKNYWRQLLNAK
LITQRKFDNLTKAERGGLSELDKAGFIKRQLVETRQITKHVAQILDSRMNTKYDENDKLI
REVKVITLKSKLVSDFRKDFQFYKVREINNYHHAHDAYLNAVVGTALIKKYPKLESEFV
YGDYKVYDVRKMIAKSEQEIGKATAKYFFYSNIMNFFKTEITLANGEIRKRPLIETNGET
GEIVWDKGRDFATVRKVLSMPQVNINKKTEVQTGGFSKESILPKRNSDKLIARKKDWDP
KKYGGFDSPTVAYSVLVVAKVEKGKSKKLKSVKELLGITIMERSSFEDNPIDFLEAKGY
KEVKKDLIIKLPKYSLFELENGRKRMLASAGELQKGNELALPSKYVNFLYLASHYEKLK
GSPEDNEQKQLFVEQHKHYLDEIIEQISEESKRVILADANLDKVLSAYNKHRDKPIREQA
ENIIHLFTLTNLGAPAAFKYFDTTIDRKRYTSTKEVLDATLIHQSITGLYETRIDLSQLGGD
KRPAATKKAGQAKKKK (SEQ ID NO: 56) Sequence for Cas9-ER50 Color code
for sequence elements: 3xFlag(MDYKDHDGDYKDHDIDYKDDDDK (SEQ ID NO:
53)) and underlined NLS from SV40 (MAPKKKRKVGIHGVPAA (SEQ ID NO:
54)) and in bold Glycine Serine linker ER50 underlined SpCas9
underlined NLS from nucleoplasmin (KRPAATKKAGQAKKKK (SEQ ID NO: 2))
and underlined
MDYKDHDGDYKDHDIDYKDDDDKMAPKKKRKVGIHGVPAADKKYSIGLDIGTNSVG
WAVITTDEYKVPSKKFKVLGNTDRHSIKKNLIGALLFDSGETAEATRLKRTARRRYTRRK
NRICYLQEIFSNEMAKVDDSFFHRLEESFLVEEDKKHERHPIFGNIVDEVAYHEKYPTIYH
LRKKLVDSTDKADLRLIYLALAHMIKFRGHFLIEGDLNPDNSDVDKLFIQLVQTYNQLFE
ENPINASGVDAKAILSARLSKSRRLENLIAQLPGEKKNGLFGNLIALSLGLTPNFKSNFDL
AEDAKLQLSKDTYDDDLDNLLAQIGDQYADLFLAAKNLSDAILLSDILRVNTEITKAPLS
ASMIKRYDEHHQDLTLLKALVRQQLPEKYKEIFFDQSKNGYAGYIDGGASQEEFYKFIK
PILEKMDGTEELLVKLNREDLLRKQRTFDNGSIPHQIHLGELHAILRRQEDFYPFLKDNR
EKIEKILTFRIPYYVGPLARGNSRFAWMTRKSEETITPWNFEEVVDKGASAQSFIERMTN
FDKNLPNEKVLPKHSLLYEYFTVYNELTKVKYVTEGMRKPAFLSGEQKKAIVDLLFKTN
RKVTVKQLKEDYFKKIECFDSVEISGVEDFRNASLGTYHDLLKIIKDKDFLDNEENEDILE
DIVLTLTLFEDREMIEERLKTYAHLFDDKVMKQLKRRRYTGWGRLSRKLINGIRDKQSG
KTILDFLKSDGFANRNFMQLIHDDSLTFKEDIQKAQVSGQGDSLHEHIANLAGSPAIKKG
ILQTVKVVDELVKVMGRHKPENIVIEMARENQTTQKGQKNSRERMKRIEEGIKELGSQI
LKEHPVENTQLQNEKLYLYYLQNGRDMYVDQELDINRLSDYDVDHIVPQSFLKDDSID
NKVLTRSDKNRGKSDNVPSEEVVKKMKNYWRQLLNAKLITQRKFDNLTKAERGGLSE
LDKAGFIKRQLVETRQITKHVAQILDSRMNTKYDENDKLIREVKVITLKSKLVSDFRKDF
QFYKVREINNYHHAHDAYLNAVVGTALIKKYPKLESEFVYGDYKVYDVRKMIAKSEQE
IGKATAKYFFYSNIMNFFKTEITLANGEIRKRPLIETNGETGEIVWDKGRDFATVRKVLS
MPQVNIVKKTEVQTGGFSKESILPKRNSDKLIARKKDWDPKKYGGFDSPTVAYSVLVV
AKVEKGKSKKLKSVKELLGITIMERSSFEKNPIDFLEAKGYKEVKKDLIIKLPKYSLFELE
NGRKRMLASAGELQKGNELALPSKYVNFLYLASHYEKLKGSPEDNEQKQLFVEQHKH
YLDEIIEQISEFSKRVILADANLDKVLSAYNKHRDKPIREQAENIIHLFTLTNLGAPAAFKY
FDTTIDRKRYTSTKEVLDATLIHQSITGLYETRIDLSQLGGDKRPAATKKAGQAKKKKS
LALSLTADQMVSALLDAEPPILYSEYDPTRPFSEASMMGLLTNLADRELVHMINWAKR
VPGFVDLALHDQVHLLECAWMEILMIGLVWRSMEHPGKLLFAPNLLLDRNQGKCVEG
GVEIFDMLLATSSRFRMMNLQGEEFVCLKSIILLNSGVYTFLSSTLKSLEEKDHIHRVLDK
ITDTLIHLMAKAGLTLQQQHQRLAQLLLILSHIRHMSSKRMEHLYSMKCKNVVPLSDLL
LEMLDAHRL (SEQ ID NO: 57) Sequence for ER50-Cas9-ER50 Color code
for sequence elements: 3xFlag(MDYKDHDGDYKDHDIDYKDDDDK (SEQ ID NO:
53)) and underlined NLS from SV40 (MAPKKKRKVGIHGVPAA (SEQ ID NO:
54)) and in bold Glycine Serine linker (GS and GSGSGS (SEQ ID NO:
55) in two parts either side of first ER50) and underlined ER50 in
two parts and underlined SpCas9 (DKK . . . GGD) NLS from
nucleoplasmin and in bold
MDYKDHDGDYKDHDIDYKDDDDKMAPKKKRKVGIHGVPAAGSSLALSLTADQMVS
ALLDAEPPILYSEYDPTRPFSEASMMGLLTNLADRELVHMINWAKRVPGFVDLALH
DQVHLLECAWMEILMIGLVWRSMEHPGKLLFAPNLLLDRNQGKCVEGGVEIFDM
LLATSSRFRMMNLQGEEFVCLKSIILLNSGVYTFLSSTLKSLEEKDHIHRVLDKITDT
LIHLMAKAGLTLQQQHQRLAQLLLILSHIRHMSSKRMEHLYSMKCKNVVPLSDLL
LEMLDAHRLGSGSGSDKKYSIGLDIGTNSVGWAVTIDEYKVPSKKFKVLGNTDRHSIK
KNLIGALLFDSGETAEATRLKRTARRRYTRRKNRICYLQEIFSNEMAKVDDSFFHRLEES
FLVEEDKKHERHPIFGNIVDEVAYHEKYPTTYHLRKKLVDSTDKADLRLIYLALAHMIKF
RGHFLIEGDLNPDNSDVDKLFIQLVQTYNQLFEENPINASGVDAKAILSARLSKSRRLEN
LIAQLPGEKKNGLFGNLIALSLGLTPNFKSNFDLAEDAKLQLSKDTYDDDLDNLLAQIGD
QYADLFLAAKNLSDAILLSDILRVNTEITKAPLSASMIKRYDEHHQDLTLLKALVRQQLP
EKYKEIFFDQSKNGYAGYIDGGASQEEFYKFIKPILEKMDGTEELLVKLNREDLLRKQRT
FDNGSIPHQIHLGELHAILRRQEDFYPFLKDNREKIEKILTFRIPYYVGPLARGNSRFAWM
TRKSEETITPWNFEEVVDKGASAQSFIERMTNFDKNLPNEKVLPKHSLLYEYFTVYNELT
KVKYVTEGMRKPAFLSGEQKKAIVDLLFKTNRKVTVKQLKEDYFKKIECFDSVEISGVE
DRFRNASLGTYHDLLKIIKDKDFLDNEENEDILEDIVLTLTLFEDREMIEERLKTYAHLFDD
KVMKQLKRRRYTGWGRLSRKLINGIRDKQSGKTILDFLKSDGFANRNFMQLIHDDSLTF
KEDIQKAQVSGQGDSLHEHIANLAGSPAIKKGILQTVKVVDELVKVMGRHKPENIVIEM
ARENQTTQKGQKNSRERMKRIEEGIKELGSQILKEHPVENTQLQNEKLYLYYLQNGRD
MYVDQELDINRLSDYDVDHIVPQSFLKDDSIDNKVLTRSDKNRGKSDNVPSEEVVKKM
KNYWRQLLNAKLITQRKFDNLTKAERGGLSELDKAGFIKRQLVETRQITKHVAQILDSR
MNTKYDENDKLIREVKVITLKSKLVSDFRKDFQFYKVREINNYHHAHDAYLNAVVGTA
LIKKYPKLESEFVYGDYKVYDVRKMIAKSEQEIGKATAKYFFYSNIMNFFKTEITLANGE
IRKRPLIETNGETGEIVWDKGRDFATVRKVLSMPQVNIVKKTEVQTGGFSKESILPKRNS
DKLIARKKDWDPKKYGGFDSPTVAYSVLVVAKVEKGKSKKLKSVKELLGITIMERSSFE
KNPIDFLEAKGYKEVKKDLIIKLPKYSLFELENGRKRMLASAGELQKGNELALPSKYVN
FLYLASHYEKLKGSPEDNEQKQLFVEQHKHYLDEIIEQISEFSKRVILADANLDKVLSAY
NKHRDKPIREQAENIIHLFTLTNLGAPAAFKYFDTTIDRKRYTSTKEVLDATLIHQSITGL
YETRIDLSQLGGDKRPAATKKAGQAKKKKSLALSLTADQMVSALLDAEPPILYSEYDP
TRPFSEASMMGLLTNLADRELVHMINWAKRVPGFVDLALHDQVHLLECAWMEILMIGL
VWRSMEHPGKLLFAPNLLLDRNQGKCVEGGVEIFDMLLATSSRFRMMNLQGEEFVCLK
SIILLNSGVYTFLSSTLKSLEEKDHIHRVLDKITDTLIHLMAKAGLTLQQQHQRLAQLLLI
LSHIRHMSSKRMEHLYSMKCKNVVPLSDLLLEMLDAHRL (SEQ ID NO: 58) Sequence
for DHFR-Cas9 Color code for sequence elements: 3xFlag and in bold
NLS from SV40 and underlined Glycine Serine linker (GS and GSGSGS
(SEQ ID NO: 55) in two parts either side of DHFR) and underlined
DHFR (ISLIAALAVDYVIGMENAMPWNLPADLAWFKRNTLNKPVIMGRHTWESIGRPLPGRKN
IILSSPQPSTDDRVTWVKSVDEAIAACGDVPEIMVIGGGRVIEQFLPKAQKLYLTHIDAEVE
GDTHFPDYEPDDWESVFSEFHDADAQNSHSYCFEILERR (SEQ ID NO: 50)) and
underlined SpCas9 (DKK . . . GGD) NLS from nucleoplasmin and in
bold MDYKDHDGDYKDHDIDYKDDDDKMAPKKKRKVGIHGVPAAGSISLIAALAVDYVIG
MENAMPWNLPADLAWFKRNTLNKPVIMGRHTWESIGRPLPGRKNIILSSQPSTDDRVT
WVKSVDEAIAACGDVPEIMVIGGGRVIEQFLPKAQKLYLTHIDAEVEGDTHFPDYEPDD
WESVFSEFHDADAQNSHSYCFEILERRGSGSGSDKKYSIGLDIGTNSVGWAVITDEYKV
PSKKFKVLGNTDRHSIKKNLIGALLFDSGETAEATRLKRTARRRYTRRKNRICYLQEIFS
NEMAKVDDSFFHRLEESFLVEEDKKHERHPIFGNIVDEVAYHEKYPIIYHLRKKLVDST
DKADLRLIYLALAHMIKFRGHFLIEGDLNPDNSDVDKLFIQLVQTYNQLFEENPINASGV
DAKAILSARLSKSRRLENLIAQLPGEKKNGLFGNLIALSLGLTPNFKSNFDLAEDAKLQL
SKDTYDDDLDNLLAQIGDQYADLFLAAKNLSDAILLSDILRVNTEITKAPLSASMIKRYD
EHHQDLTLLKALVRQQLPEKYKEIFFDQSKNGYAGYIDGGASQEEFYKFIKPILEKMDGT
EELLVKLNREDLLRKQRTFDNGSIPHQIHLGELHAILRRQEDFYPFLKDNREKIEKILTFRI
PYYVGPLARGNSRFAWMTRKSEETTTPWNFEEVVDKGASAQSFIERMTNFDKNLPNEK
VLPKHSLLYEYFTVYNELTKVKYVTEGMRKPAFLSGEQKKAIVDLLFKTNRKVTVKQL
KEDYFKKIECFDSVEISGVEDRFNASLGTYHDLLKIIKDKDFLDNEENEDILEDIVLTLTLF
EDREMIEERLKTYAHLFDDKVMKQLKRRRYTGWGRLSRKLINGIRDKQSGKTILDFLKS
DGFANRNFMQLIHDDSLTFKEDIQKAQVSGQGDSLHEHIANLAGSPAIKKGILQTVKVV
DELVKVMGRHKPENIVIEMARENQTTQKGQKNSRERMKRIEEGIKELGSQILKEHPVEN
TQLQNEKLYLYYLQNGRDMYVDQELDINRLSDYDVDHIVPQSFLKDDSIDNKVLTRSD
KNRGKSDNVPSEEVVKKMKNYWRQLLNAKLITQRKFDNLTKAERGGLSELDKAGFIKR
QLVETRQITKHVAQILDSRMNTKYDENDKLIREVKVITLKSKLVSDFRKDFQFYKVREIN
NYHHAHDAYLNAVVGTALIKKYPKLESEFVYGDYKVYDVRKMIAKSEQEIGKATAKYF
FYSNIMNFFKTEITLANGEIRKRPLIETNGETGEIVWDKGRDFATVRKVLSMPQVNIVKK
TEVQTGGFSKESILPKRNSDKLIARKKDWDPKKYGGFDSPTVAYSVLVVAKVEKGKSK
KLKSVKELLGITIMERSSFEKNPIDFLEAKGYKEVKKDLIIKLPKYSLFELENGRKRMLAS
AGELQKGNELALPSKYVNFLYLASHYEKLKGSPEDNEQKQLFVEQHKHYLDEIIEQISEF
SKRVILADANLDKVLSAYNKHRDKPIREQAENIIHLFTLTNLGAPAAFKYFDTTIDRKRY
TSTKEVLDATLIHQSITGLYETRIDLSQLGGDKRPAATKKAGQAKKKK (SEQ ID NO: 59)
Sequence for Cas9-DHFR Color code for sequence elements: 3xFlag and
underlined NLS from SV40 and in bold Glycine Serine linker and
underlined DHFR and in bold SpCas9 (DKK . . . GGD) NLS from
nucleoplasmin (KRPAATKKAGQAKKKK (SEQ ID NO: 2)) and underlined and
in bold MDYKDHDGDYKDHDIDYKDDDDKMAPKKKRKVGIHGVPAADKKYSIGLDIGTNSVG
WAVITDEYKVPSKKFKVLGNTDRHSIKKNLIGALLFDSGETAEATRLKRTARRRYTRRK
NRICYLQEIFSNEMAKVDDSFFHRLEESFLVEEDKKHERHPIFGNIVDEVAYHEKYPITYH
LRKKLVDSTDKADLRLIYLALAHMIKFRGHFLIEGDLNPDNSDVDKLFIQLVQTYNQLFE
ENPINASGVDAKAILSARLSKSRRLENLIAQLPGEKKNGLFGNLIALSLGLTPNFKSNFDL
AEDAKLQLSKDTYDDDLDNLLAQIGDQYADLFLAAKNLSDAILLSDILRVNTEITKAPLS
ASMIKRYDEHHQDLTLLKALVRQQLPEKYKEIFFDQSKNGYAGYIDGGASQEEFYKFIK
PILEKMDGTEELLVKLNREDLLRKQRTFDNGSIPHQIHLGELHAILRRQEDFYPFLKDNR
EKIEKILTFRIPYYVGPLARGNSRFAWMTRKSEETITPWNFEEVVDKGASAQSFIERMTN
FDKNLPNEKVLPKHSLLYEYFTVYNELTKVKYVTEGMRKPAFLSGEQKKAIVDLLFKTN
RKVTVKQLKEDYFKKIECFDSVEISGVEDRFNASLGTYHDLLKIIKDKDFLDNEENEDILE
DIVLTLTLFEDREMIEERLKTYAHLFDDKVMKQLKRRRYTGWGRLSRKLINGIRDKQSG
KTILDFLKSDGFANRNFMQLIHDDSLTFKEDIQKAQVSGQGDSLHEHIANLAGSPAIKKG
ILQTVKVVDELVKVMGRHKPENIVIEMARENQTTQKGQKNSRERMKRIEEGIKELGSQI
LKEHPVENTQLQNEKLYLYYLQNGRDMYVDQELDINRLSDYDVDHIVPQSFLKDDSID
NKVLTRSKDNRGKSDNVPSEEVVKKMKNYWRQLLNAKLITQRKFDNLTKAERGGLSE
LDKAGFIKRQLVETRQITKHVAQILDSRMNTKYDENDKLIREVKVITLKSKLVSDFRKDF
QFYKVREINNYHHAHDAYLNAVVGTALIKKYPKLESEFVYGDYKVYDVRKMIAKSEQE
IGKATAKYFFYSNIMNFFKTEITLANGEIRKRPLIETNGETGEIVWDKGRDFATVRKVLS
MPQVNIVKKTEVQTGGFSKESILPKRNSDKLIARKKDWDPKKYGGFDSPTVAYSVLVV
AKVEKGKSKKLKSVKELLGITIMERSSFEKNPIDFLEAKGYKEVKKDLIIKLPKYSLFELE
NGRKRMLASAGELQKGNELALPSKYVNFLYLASHYEKLKGSPEDNEQKQLFVEQHKH
YLDEIIEQISEFSKRVILADANLDKVLSAYNKHRDKPIREQAENIIHLFTLTNLGAPAAFKY
FDTTIDRKRYTSTKEVLDATLIHQSITGLYETRIDLSQLGGDKRPAATKKAGQAKKKKG
SISLIAALAVDHVIGMETVMPWNLPADLAWFKRNTLNKPVIMGRHTWESIGRPLPG
RKNIILSSQPSTDDRVTWVKSVDEAIAACGDVPEIMVIGGGRVYEQFLPKAQKLYLT
HIDAEVEGDTHFPDYEPDDWESVFSEFHDADAQNSHSYCFEILERR (SEQ ID NO: 60)
Sequence for DHFR-Cas9-DHFR Color code for sequence elements:
3xFlag and underlined NLS from SV40 and in bold Glycine Serine
linker (GS and GSGSGS (SEQ ID NO: 55) in two parts either side of
first DHFR) and underlined DHFR and in bold SpCas9 (DKK . . . GGD)
NLS from nucleoplasmin and underlined and in bold
MDYKDHDGDYKDHDIDYKDDDDKMAPKKKRKVGIHGVPAAGSISLIAALAVDYVIG
MENAMPWNLPADLAWFKRNTLNKPVIMGRHTWESIGRPLPGRKNIILSSQPSTDD
RVTWVKSVDEAIAACGDVPEIMVIGGGRVIEQFLPKAQKLYLTHIDAEVEGDTHFP
DYEPDDWESVFSEFHDADAQNSHSYCFEILERRGSGSGSDKKYSIGLDIGTNSVGWAV
ITDEYKVPSKKFKVLGNTDRHSIKKNLIGALLFDSGETAEATRLKRTARRRYTRRKNRIC
YLQEIFSNEMAKVDDSFFHRLEESFLVEEDKKHERHPIFGNIVDEVAYHEKYPTIYHLRK
KLVDSTDKADLRIYLALAHMIKFRGHFLIEGDLNPDNSEDVDKLFIQLVQTYNQLFEENP
INASGVDAKAILSARLSKSRRLENLIAQLPGEKKNGLFGNLIALSLGLTPNFKSNFDLAED
AKLQLSKDTYDDDLDNLLAQIGDQYADLFLAAKNLSDAILLSDILRVNTEITKAPLSASM
IKRYDEHHQDLTLLKALVRQQLPEKYKEIFFDQSKNGYAGYIDGGASQEEFYKFIKPILE
KMDGTEELLVKLNREDLLRKQRTFDNGSIPHQIHLGELHAILRRQEDFYPFLKDNREKIE
KILTFRIPYYVGPLARGNSRFAWMTRKSEETITPWNFEEVVDKGASAQSFIERMTNFDKN
LPNEKVLPKHSLLYEYFTVYNELTKVKYVTEGMRKPAFLSGEQKKAIVDLLFKTNRKVT
VKQLKEDYFKKIECFDSVEISGVEDRFNASLGTYHDLLKIIKDKDFLDNEENEDILEDIVL
TLTLFEDREMIEERLKTYAHLFDDKVMKQLKRRRYTGWGRLSRKLINGIRDKQSGKTIL
DFLKSDGFANRNFMQLIHDDSLTFKEDIQKAQVSGQGDSLHEHIANLAGSPAIKKGILQT
VKVVDELVKVMGRHKPENIVIEMARENQTIQKGQKNSRERMKRIEEGIKELGSQILKEH
PVENTQLQNEKLYLYYLQNGRDMYVDQELDINRLSDYDVDHIVPQSFLKDDSIDNKVL
TRSDKNRGKSDNVPSEEVVKKMKNYWRQLLNAKLITQRKFDNLTKAERGGLSELDKA
GFIKRQLVETRQITKHVAQILDSRMNTKYDENDKLIREVKVITLKSKLVSDFRKDFQFYK
VREINNYHHAHDAYLNAVVGTALIKKYPKLESEFVYGDYKVYDVRKMIAKSEQEIGKA
TAKYFFYSNIMNFFKTEITLANGEIRKRPLIETNGETGEIVWDKGRDFATVRKVLSMPQV
NIVKKTEVQTGGFSKESILPKRNSDKLLIARKKDWDPKKYGGFDSPTVAYSVLVVAKVEK
GKSKKLKSVKELLGITIMERSSFEKNPIDFLEAKGYKEVKKDLIIKLPKYSLFELENGRKR
MLASAGELQKGNELALPSKYVNFLYLASHYEKLKGSPEDNEQKQLFVEQHKHYLDEIIE
QISEFSKRVILADANLDKVLSAYNKHRDKPIREQAENIIHLFTLTNLGAPAAFKYFDTTID
RKRYTSTKEVLDATLIHQSITGLYETRIDLSQLGGDKRPAATKKAGQAKKKKISLIAAL
AVDHVIGMETVMPWNLPADLAWFKRNTLNKPVIMGRHTWESIGRPLPGRKNIILS
SQPSTDDRVTWVKSVDEAIAACGDVPEIMVIGGGRVYEQFLPKAQKLYLTHIDAEV
EGDTHFPDYEPDDWESVFSEFHDADAQNSHSYCFEILERR (SEQ ID NO: 61)
[0856] Cell culture and drug treatment: Human embryonic kidney
293FT (HEK293FT) cell line (Life Technologies) were maintained in
Dulbecco's modified Eagle's Medium (DMEM) supplemented with 10% FBS
(HyClone), 2 mM GlutaMAX (Life Technologies), 100 U/ml penicillin,
and 100 .mu.g/ml streptomycin at 37.degree. C. with 5% CO2
incubation. HEK293FT cells were seeded onto 24-well plates
(Corning) 24 h before transfection. Cells were transfected using
Lipofectamine 2000 (Life Technologies) at 80-90% confluency
following the manufacturer's recommended protocol. For each well of
a 24-well plate, a total of 500 ng DNA was used. 400 ng of DD-Cas9
plasmid with 100 ng PCR amplified U6-sgRNA targeting EMX1 was
transfected. Cells were treated with 4HT, CMP8 or TMP ligands at
the time of transfection. Ligands where devolved in DMSO and
diluted to final concentration in culture medium. Cells were
harvested three days after transfection.
[0857] Deep sequencing to assess targeting specificity: Genomic DNA
was extracted using the QuickExtract DNA Extraction Solution
(Epicentre) following the manufacturer's protocol. Briefly,
pelleted cells were resuspended in QuickExtract solution and
incubated at 65.degree. C. for 15 min, 68.degree. C. for 15 min,
and 98.degree. C. for 10 min. The genomic region flanking the
CRISPR target site for EMX1 or OTs were amplified by a fusion PCR
method to attach the Illumina P5 adapters as well as unique
sample-specific barcodes to the target amplicons PCR products were
purified by gel-extraction using QiaQuick Spin Column (Qiagen)
following the manufacturer's recommended protocol. Barcoded and
purified DNA samples were quantified by Quant-iT PicoGreen dsDNA
Assay Kit or Qubit 2.0 Fluorometer (Life Technologies) and pooled
in an equimolar ratio. Sequencing libraries were then sequenced
with the Illumina MiSeq Personal Sequencer (Life Technologies).
[0858] Sequencing data analysis and indel detection: MiSeq reads
were filtered by requiring an average Phred quality (Q score) of at
least 30, as well as perfect sequence matches to barcodes and
amplicon forward primers. Reads from on- and off-target loci were
analyzed by performing Ratcliff-Obershelp string comparison, as
implemented in the Python difflib module, against loci sequences
that included 30 nt upstream and downstream of the target site (a
total of 80 bp). The resulting edit operations were parsed, and
reads were counted as indels if insertion or deletion operations
were found. Analyzed target regions were discarded if part of their
alignment fell outside of the MiSeq read itself or if more than
five bases were uncalled.
[0859] FIG. 1 shows indels detected by deep sequencing in HEK293FT
cells transfected with 400 ng ER50-Cas9, Cas9-ER50 or
ER50-Cas9-ER50 and 100 ng PCR amplified U6-sgRNA targeting EMX1.
ER50 was stabilized with either 4-hydroxytamoxifen (4HT) or CMP8.
Two different concentrations were tested for each ligand. FIG. 2
shows indels detected by deep sequencing in HEK293FT cells
transfected with 400 ng DHFR-Cas9, Cas9-DHFR or DHFR-Cas9-DHFR and
100 ng PCR amplified U6-sgRNA targeting EMX1. ER50 was stabilized
with trimethoprim (TMP). Two different concentrations were tested.
FIG. 3 shows when ER50 or DHFR was fused to the C-term of
FKBP(C)Cas9-2.times.NLS, indels were detected by deep sequencing in
HEK293FT cells transfected with 200 ng FRB(N)Cas9-NLS,
FRB(N)Cas9-noNLS or FRB(N)Cas9-NES and with 200 ng
FKBP(C)Cas9-2.times.NLS-DD and 100 ng PCR amplified U6-sgRNA
targeting EMX1. ER50 fusions were treated with either 10 uM 4HT or
1.5 uM CMP8. DHFR fusions were treated with 10 uM TMP.
Example 2: Construction and Testing of DD-Cas9 Using Cas9
[0860] Example 1 was repeated with a Cas9 from Staphylococcus
aureus. The results with activation of transcription of the ASCL1
gene into ASCL1 mRNA are shown in FIG. 4. The DD used are DHFR and
ER50, as before. The stabilizing ligands are TMP and 4HT,
respectively, as before. Significant reductions in transcription
are seen in the absence of each stabilizing ligand compared to in
the presence of each ligand (for the corresponding DD). This
provides further and useful validation of the results in Example 1
in an ortholog of SpCas9, SaCas9.
Example 3
[0861] Joung and co-workers have reported (High-frequency
off-target mutagenesis induced by CRISPR-Cas nucleases in human
cells. Fu, Y.; Foden, J. A.; Khayter, C.; Maeder, M. L.; Reyon, D.;
Joung, J. K.; Sander, J. D. Nat Biotechnol 2013, 31,
822-6.PMC3773023) a U2OS.EGFP cell line wherein knockdown of EGFP
gene by SpCas9 leads to loss of EGFP fluorescence. By quantifying
the percentage of EGFP positive cells using flow cytometry, one can
estimate SpCas9 activity. The low throughput, flow
cytometry-readout was replaced with a high-throughput readout using
a high-content, automated microscope imaging. This assay can be run
in a 96-well format and the data can be analyzed by a
high-throughput image analysis platform, called ImageExpress. Using
this optimized protocol, dosage control of Cas9 activity was
demonstrated using both DHFR and ER50 destabilized domains. The
amount of plasmid and DD ligand used in the experiments are
indicated in FIGS. 6 and 7.
[0862] In one case, DHFR DDs were fused to SpCas9. The
DHFR-SpCas9-DHFR (DSPD) fusion protein was provided to an
EGFP-producing cell line with a guide RNA targeted to EGFP gene.
FIG. 6 shows EGFP fluorescence was reduced to about 60-80% of the
untreated level, depending on the dose of DPSD. Addition of the
DHFR ligand trimethoprim (TMP) increased the effect of the DSPD
fusion protein in a dose-dependent manner. This indicates that by
binding to DHFR DD, TMP stabilized the DSPD fusion protein, leading
to increased SpCas9 cleavage of EGFP.
[0863] In the second case, ER50 DDs were fused to SpCas9. The
ER50-SpCas9-ER50 (ESPE) fusion protein was provided to an
EGFP-producing cell line with a guide RNA targeted to EGFP gene.
FIG. 6 shows EGFP fluorescence was reduced to about 70-80% of the
untreated level, depending on the dose of EPSE. Addition of the
ER50 ligand 4HT increased the effect of the ESPE fusion protein in
a dose-dependent manner. This indicates that by binding to ER50 DD,
4HT stabilized the ESPE fusion protein, leading to increased SpCas9
cleavage of EGFP.
[0864] The invention is further described by the following numbered
paragraphs:
[0865] 1. A non-naturally occurring or engineered CRISPR Cas9
associated with or fused to at least one destabilization domain
(DD)
[0866] 2. The Cas9 of numbered paragraph 1, which comprises an Sp
Cas9.
[0867] 3. The Cas9 of numbered paragraph 1, which comprises an Sa
Cas9.
[0868] 4. The Cas9 of numbered paragraph 1, which comprises an St
or Fn Cas9.
[0869] 5. The Cas9 of any preceding numbered paragraph, which
comprises a Rec2 or HD2 truncation.
[0870] 6. The Cas9 of numbered paragraph 5 wherein the truncation
comprises removal or replacement with a linker.
[0871] 7. The Cas9 of numbered paragraph 6 wherein the linker
comprises a branch or otherwise allows for tethering of the DD
and/or a functional domain.
[0872] 8. The Cas9 of any preceding numbered paragraph, wherein the
DD is associated with the CRISPR Cas9 by fusion with said CRISPR
Cas9.
[0873] 9. The Cas9 of numbered paragraph 8, which comprises at
least one DD fused to the N-terminus of the CRISPR Cas9.
[0874] 10. The Cas9 of numbered paragraph 8, which comprises at
least one DD fused to the C-terminus of the CRISPR Cas9.
[0875] 11. The Cas9 of numbered paragraph 8, comprising at least
two DDs and wherein a first DD is fused to the N-terminus of the
CRISPR Cas9 and a second DD is fused to the C-terminus of the
CRISPR Cas9, the first and second DDs being the same or
different.
[0876] 12. The Cas9 of any of numbered paragraphs 8-11, wherein the
fusion comprises a linker between the DD and the CRISPR Cas9.
[0877] 13. The Cas9 of numbered paragraph 12, wherein the linker
comprises, consists essentially of or consists of: GlySer linker;
or a localization signal.
[0878] 14. The Cas9 of any preceding numbered paragraph, further
comprising at least one Nuclear Export Signal (NES).
[0879] 15. The Cas9 of numbered paragraph 14, comprising two or
more NESs.
[0880] 16. The Cas9 of any preceding numbered paragraph, further
comprising at least one Nuclear Localization Signal (NLS).
[0881] 17. The Cas9 of any preceding numbered paragraph, wherein
one of the at least one DD comprises ER50.
[0882] 18. The Cas9 of any preceding numbered paragraph, wherein
one of the at least one DDs is DHFR50.
[0883] 19. The Cas9 of any preceding numbered paragraph which
comprises at least one mutation.
[0884] 20. The Cas9 of numbered paragraph 19 wherein the Cas9
comprises a nickase.
[0885] 21. The Cas9 of numbered paragraph 20 wherein the Cas9
comprises Staphylococcus aureus Cas9 (SaCas9) and the mutation
comprises N580A.
[0886] 22. The Cas9 of numbered paragraph 19 wherein the Cas9 has
substantially no nuclease activity due to the mutation(s).
[0887] 23. The Cas9 of any one of numbered paragraphs 1-18, wherein
the Cas9 comprises a split Cas9.
[0888] 24. The Cas9 of any one of numbered paragraphs 1-18, wherein
the Cas9 comprises a functional domain.
[0889] 25. A polynucleotide encoding the CRISPR Cas9 and associated
DD of any one of the preceding numbered paragraphs.
[0890] 26. The polynucleotide of numbered paragraph 25, wherein the
encoded CRISPR Cas9 and associated DD are operably linked to a
first regulatory element.
[0891] 27. The polynucleotide of numbered paragraph 25 or 26,
wherein a DD is encoded and is operably linked to a second
regulatory element.
[0892] 28. The polynucleotide of any of numbered paragraphs 25-27,
wherein the first regulatory element comprises a promoter and may
optionally comprises an enhancer.
[0893] 29. The polynucleotide of any of numbered paragraphs 25-28,
wherein the second regulatory element comprises a promoter and may
optionally comprises an enhancer.
[0894] 30. The polynucleotide of any of numbered paragraphs 25-29,
wherein the first regulatory element comprises an early
promoter.
[0895] 31. The polynucleotide of any of numbered paragraphs 25-30,
wherein the second regulatory element is a late promoter.
[0896] 32. The polynucleotide of any of numbered paragraphs 25-31,
wherein the second regulatory element comprises an inducible
control element, optionally the tet system, or a repressible
control element, optionally the tetr system.
[0897] 33. A vector(s) comprising the polynucleotide(s) of any of
numbered paragraphs 25-32.
[0898] 34. The vector(s) of numbered paragraph 33, comprising one
or more plasmid or viral vector.
[0899] 35. A cell or cell line or non-human animal, or progeny
thereof, modified so as to contain an Cas9 of any one of numbered
paragraphs 1-24 or a polynucleotide of numbered paragraphs 25-32 or
a vector(s) of a numbered paragraph 33 or 34.
[0900] 36. The non-human animal of numbered paragraph 35, which
constitutively expresses the CRISPR Cas9-DD fusion.
[0901] 37. The non-human animal of numbered paragraph 36, wherein
the non-human animal is a mouse.
[0902] 38. Progeny of the cell or cell line or non-human animal of
any of numbered paragraphs 35-38.
[0903] 39. A DD-CRISPR-Cas System comprising a Cas9 of any one of
numbered paragraphs 1-24, a polynucleotide of any one of numbered
paragraphs 25-32, or a vector of any one of numbered paragraphs 33
or 34.
[0904] 40. A method of controlled targeting of a polynucleotide of
interest in a cell comprising having a DD-CRISPR-Cas9 of numbered
paragraph 39 present in the cell whereby guide RNA of the DD-CRISPR
Cas9 targets the polynucleotide of interest; and having, in a
controlled manner, a stabilizing ligand as to the DD present in the
cell.
[0905] 41. A method of treatment of a subject in need thereof
comprising administering a DD-CRISPR-Cas9 of numbered paragraph 39
to the subject whereby it is present in cell(s) of the subject
whereby guide RNA of the DD-CRISPR Cas targets a polynucleotide of
interest involved in a condition of the subject, the targeting of
which results in a treatment therefor; and having, in a controlled
manner, a stabilizing ligand as to the DD present in cell(s) of the
subject.
[0906] 43. A CRISPR-Cas9 complex comprising the Cas9 according to
or as defined in any one of the preceding numbered paragraphs, a
guide RNA and nucleic acid target.
[0907] While preferred embodiments of the present invention have
been shown and described herein, it will be obvious to those
skilled in the art that such embodiments are provided by way of
example only. Numerous variations, changes, and substitutions will
now occur to those skilled in the art without departing from the
invention. It should be understood that various alternatives to the
embodiments of the invention described herein may be employed in
practicing the invention. It is intended that the following claims
define the scope of the invention and that methods and structures
within the scope of these claims and their equivalents be covered
thereby.
Sequence CWU 1
1
6917PRTSimian virus 40 1Pro Lys Lys Lys Arg Lys Val 1 5
216PRTUnknownsource/note="Description of Unknown Nucleoplasmin
bipartite NLS sequence" 2Lys Arg Pro Ala Ala Thr Lys Lys Ala Gly
Gln Ala Lys Lys Lys Lys 1 5 10 15
39PRTUnknownsource/note="Description of Unknown C-myc NLS sequence"
3Pro Ala Ala Lys Arg Val Lys Leu Asp 1 5
411PRTUnknownsource/note="Description of Unknown C-myc NLS
sequence" 4Arg Gln Arg Arg Asn Glu Leu Lys Arg Ser Pro 1 5 10
538PRTHomo sapiens 5Asn Gln Ser Ser Asn Phe Gly Pro Met Lys Gly Gly
Asn Phe Gly Gly 1 5 10 15 Arg Ser Ser Gly Pro Tyr Gly Gly Gly Gly
Gln Tyr Phe Ala Lys Pro 20 25 30 Arg Asn Gln Gly Gly Tyr 35
642PRTUnknownsource/note="Description of Unknown IBB domain from
importin-alpha sequence" 6Arg Met Arg Ile Glx Phe Lys Asn Lys Gly
Lys Asp Thr Ala Glu Leu 1 5 10 15 Arg Arg Arg Arg Val Glu Val Ser
Val Glu Leu Arg Lys Ala Lys Lys 20 25 30 Asp Glu Gln Ile Leu Lys
Arg Arg Asn Val 35 40 78PRTUnknownsource/note="Description of
Unknown Myoma T Protein sequence" 7Val Ser Arg Lys Arg Pro Arg Pro
1 5 88PRTUnknownsource/note="Description of Unknown Myoma T Protein
sequence" 8Pro Pro Lys Lys Ala Arg Glu Asp 1 5 98PRTHomo sapiens
9Pro Gln Pro Lys Lys Lys Pro Leu 1 5 1012PRTMus musculus 10Ser Ala
Leu Ile Lys Lys Lys Lys Lys Met Ala Pro 1 5 10 115PRTInfluenza
virus 11Asp Arg Leu Arg Arg 1 5 127PRTInfluenza virus 12Pro Lys Gln
Lys Lys Arg Lys 1 5 1310PRTHepatitis delta virus 13Arg Lys Leu Lys
Lys Lys Ile Lys Lys Leu 1 5 10 1410PRTMus musculus 14Arg Glu Lys
Lys Lys Phe Leu Lys Arg Arg 1 5 10 1520PRTHomo sapiens 15Lys Arg
Lys Gly Asp Glu Val Asp Gly Val Asp Glu Val Ala Lys Lys 1 5 10 15
Lys Ser Lys Lys 20 1617PRTHomo sapiens 16Arg Lys Cys Leu Gln Ala
Gly Met Asn Leu Glu Ala Arg Lys Thr Lys 1 5 10 15 Lys
1727DNAArtificial Sequencesource/note="Description of Artificial
Sequence Synthetic oligonucleotide"modified_base(1)..(20)a, c, t or
gmodified_base(21)..(22)a, c, t, g, unknown or other 17nnnnnnnnnn
nnnnnnnnnn nnagaaw 271819DNAArtificial
Sequencesource/note="Description of Artificial Sequence Synthetic
oligonucleotide"modified_base(1)..(12)a, c, t or
gmodified_base(13)..(14)a, c, t, g, unknown or other 18nnnnnnnnnn
nnnnagaaw 191927DNAArtificial Sequencesource/note="Description of
Artificial Sequence Synthetic
oligonucleotide"modified_base(1)..(20)a, c, t or
gmodified_base(21)..(22)a, c, t, g, unknown or other 19nnnnnnnnnn
nnnnnnnnnn nnagaaw 272018DNAArtificial
Sequencesource/note="Description of Artificial Sequence Synthetic
oligonucleotide"modified_base(1)..(11)a, c, t or
gmodified_base(12)..(13)a, c, t, g, unknown or other 20nnnnnnnnnn
nnnagaaw 1821137DNAArtificial Sequencesource/note="Description of
Artificial Sequence Synthetic
polynucleotide"modified_base(1)..(20)a, c, t, g, unknown or other
21nnnnnnnnnn nnnnnnnnnn gtttttgtac tctcaagatt tagaaataaa tcttgcagaa
60gctacaaaga taaggcttca tgccgaaatc aacaccctgt cattttatgg cagggtgttt
120tcgttattta atttttt 13722123DNAArtificial
Sequencesource/note="Description of Artificial Sequence Synthetic
polynucleotide"modified_base(1)..(20)a, c, t, g, unknown or other
22nnnnnnnnnn nnnnnnnnnn gtttttgtac tctcagaaat gcagaagcta caaagataag
60gcttcatgcc gaaatcaaca ccctgtcatt ttatggcagg gtgttttcgt tatttaattt
120ttt 12323110DNAArtificial Sequencesource/note="Description of
Artificial Sequence Synthetic
polynucleotide"modified_base(1)..(20)a, c, t, g, unknown or other
23nnnnnnnnnn nnnnnnnnnn gtttttgtac tctcagaaat gcagaagcta caaagataag
60gcttcatgcc gaaatcaaca ccctgtcatt ttatggcagg gtgttttttt
11024102DNAArtificial Sequencesource/note="Description of
Artificial Sequence Synthetic
polynucleotide"modified_base(1)..(20)a, c, t, g, unknown or other
24nnnnnnnnnn nnnnnnnnnn gttttagagc tagaaatagc aagttaaaat aaggctagtc
60cgttatcaac ttgaaaaagt ggcaccgagt cggtgctttt tt
1022588DNAArtificial Sequencesource/note="Description of Artificial
Sequence Synthetic oligonucleotide"modified_base(1)..(20)a, c, t,
g, unknown or other 25nnnnnnnnnn nnnnnnnnnn gttttagagc tagaaatagc
aagttaaaat aaggctagtc 60cgttatcaac ttgaaaaagt gttttttt
882676DNAArtificial Sequencesource/note="Description of Artificial
Sequence Synthetic oligonucleotide"modified_base(1)..(20)a, c, t,
g, unknown or other 26nnnnnnnnnn nnnnnnnnnn gttttagagc tagaaatagc
aagttaaaat aaggctagtc 60cgttatcatt tttttt 762715PRTArtificial
Sequencesource/note="Description of Artificial Sequence Synthetic
peptide" 27Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly
Ser 1 5 10 15 284PRTArtificial Sequencesource/note="Description of
Artificial Sequence Synthetic peptide" 28Gly Gly Gly Ser 1
2912PRTArtificial Sequencesource/note="Description of Artificial
Sequence Synthetic peptide" 29Gly Gly Gly Ser Gly Gly Gly Ser Gly
Gly Gly Ser 1 5 10 307PRTArtificial
Sequencesource/note="Description of Artificial Sequence Synthetic
peptide" 30Ala Glu Ala Ala Ala Lys Ala 1 5 3130PRTArtificial
Sequencesource/note="Description of Artificial Sequence Synthetic
polypeptide" 31Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly
Gly Ser Gly 1 5 10 15 Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly
Gly Gly Ser 20 25 30 3245PRTArtificial
Sequencesource/note="Description of Artificial Sequence Synthetic
polypeptide" 32Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly
Gly Ser Gly 1 5 10 15 Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly
Gly Gly Ser Gly Gly 20 25 30 Gly Gly Ser Gly Gly Gly Gly Ser Gly
Gly Gly Gly Ser 35 40 45 3360PRTArtificial
Sequencesource/note="Description of Artificial Sequence Synthetic
polypeptide" 33Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly
Gly Ser Gly 1 5 10 15 Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly
Gly Gly Ser Gly Gly 20 25 30 Gly Gly Ser Gly Gly Gly Gly Ser Gly
Gly Gly Gly Ser Gly Gly Gly 35 40 45 Gly Ser Gly Gly Gly Gly Ser
Gly Gly Gly Gly Ser 50 55 60 345PRTArtificial
Sequencesource/note="Description of Artificial Sequence Synthetic
peptide" 34Gly Gly Gly Gly Ser 1 5 3510PRTArtificial
Sequencesource/note="Description of Artificial Sequence Synthetic
peptide" 35Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser 1 5 10
3620PRTArtificial Sequencesource/note="Description of Artificial
Sequence Synthetic peptide" 36Gly Gly Gly Gly Ser Gly Gly Gly Gly
Ser Gly Gly Gly Gly Ser Gly 1 5 10 15 Gly Gly Gly Ser 20
3725PRTArtificial Sequencesource/note="Description of Artificial
Sequence Synthetic peptide" 37Gly Gly Gly Gly Ser Gly Gly Gly Gly
Ser Gly Gly Gly Gly Ser Gly 1 5 10 15 Gly Gly Gly Ser Gly Gly Gly
Gly Ser 20 25 3835PRTArtificial Sequencesource/note="Description of
Artificial Sequence Synthetic polypeptide" 38Gly Gly Gly Gly Ser
Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly 1 5 10 15 Gly Gly Gly
Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly 20 25 30 Gly
Gly Ser 35 3940PRTArtificial Sequencesource/note="Description of
Artificial Sequence Synthetic polypeptide" 39Gly Gly Gly Gly Ser
Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly 1 5 10 15 Gly Gly Gly
Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly 20 25 30 Gly
Gly Ser Gly Gly Gly Gly Ser 35 40 4050PRTArtificial
Sequencesource/note="Description of Artificial Sequence Synthetic
polypeptide" 40Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly
Gly Ser Gly 1 5 10 15 Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly
Gly Gly Ser Gly Gly 20 25 30 Gly Gly Ser Gly Gly Gly Gly Ser Gly
Gly Gly Gly Ser Gly Gly Gly 35 40 45 Gly Ser 50 4155PRTArtificial
Sequencesource/note="Description of Artificial Sequence Synthetic
polypeptide" 41Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly
Gly Ser Gly 1 5 10 15 Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly
Gly Gly Ser Gly Gly 20 25 30 Gly Gly Ser Gly Gly Gly Gly Ser Gly
Gly Gly Gly Ser Gly Gly Gly 35 40 45 Gly Ser Gly Gly Gly Gly Ser 50
55 42414DNAStreptococcus pyogenes 42ctgaaccccg acaacagcga
cgtggacaag ctgttcatcc agctggtgca gacctacaac 60cagctgttcg aggaaaaccc
catcaacgcc agcggcgtgg acgccaaggc catcctgtct 120gccagactga
gcaagagcag acggctggaa aatctgatcg cccagctgcc cggcgagaag
180aagaatggcc tgttcggaaa cctgattgcc ctgagcctgg gcctgacccc
caacttcaag 240agcaacttcg acctggccga ggatgccaaa ctgcagctga
gcaaggacac ctacgacgac 300gacctggaca acctgctggc ccagatcggc
gaccagtacg ccgacctgtt tctggccgcc 360aagaacctgt ccgacgccat
cctgctgagc gacatcctga gagtgaacac cgag 414434PRTArtificial
Sequencesource/note="Description of Artificial Sequence Synthetic
peptide" 43Gly Gly Ser Gly 1 4412PRTArtificial
Sequencesource/note="Description of Artificial Sequence Synthetic
peptide"MOD_RES(2)..(2)AhxMOD_RES(5)..(5)AhxMOD_RES(8)..(8)AhxMOD_RES(11)-
..(11)Ahx 44Arg Xaa Arg Arg Xaa Arg Arg Xaa Arg Arg Xaa Arg 1 5 10
4520DNAHomo sapiens 45gagtccgagc agaagaagaa 204620DNAHomo sapiens
46gagtcctagc aggagaagaa 204720DNAHomo sapiens 47gagtctaagc
agaagaagaa 204812RNAArtificial Sequencesource/note="Description of
Artificial Sequence Synthetic oligonucleotide" 48guuuuagagc ua
12499PRTUnknownsource/note="Description of Unknown "LAGLIDADG"
family motif peptide" 49Leu Ala Gly Leu Ile Asp Ala Asp Gly 1 5
50158PRTArtificial Sequencesource/note="Description of Artificial
Sequence Synthetic polypeptide" 50Ile Ser Leu Ile Ala Ala Leu Ala
Val Asp Tyr Val Ile Gly Met Glu 1 5 10 15 Asn Ala Met Pro Trp Asn
Leu Pro Ala Asp Leu Ala Trp Phe Lys Arg 20 25 30 Asn Thr Leu Asn
Lys Pro Val Ile Met Gly Arg His Thr Trp Glu Ser 35 40 45 Ile Gly
Arg Pro Leu Pro Gly Arg Lys Asn Ile Ile Leu Ser Ser Gln 50 55 60
Pro Ser Thr Asp Asp Arg Val Thr Trp Val Lys Ser Val Asp Glu Ala 65
70 75 80 Ile Ala Ala Cys Gly Asp Val Pro Glu Ile Met Val Ile Gly
Gly Gly 85 90 95 Arg Val Ile Glu Gln Phe Leu Pro Lys Ala Gln Lys
Leu Tyr Leu Thr 100 105 110 His Ile Asp Ala Glu Val Glu Gly Asp Thr
His Phe Pro Asp Tyr Glu 115 120 125 Pro Asp Asp Trp Glu Ser Val Phe
Ser Glu Phe His Asp Ala Asp Ala 130 135 140 Gln Asn Ser His Ser Tyr
Cys Phe Glu Ile Leu Glu Arg Arg 145 150 155 51158PRTArtificial
Sequencesource/note="Description of Artificial Sequence Synthetic
polypeptide" 51Ile Ser Leu Ile Ala Ala Leu Ala Val Asp His Val Ile
Gly Met Glu 1 5 10 15 Thr Val Met Pro Trp Asn Leu Pro Ala Asp Leu
Ala Trp Phe Lys Arg 20 25 30 Asn Thr Leu Asn Lys Pro Val Ile Met
Gly Arg His Thr Trp Glu Ser 35 40 45 Ile Gly Arg Pro Leu Pro Gly
Arg Lys Asn Ile Ile Leu Ser Ser Gln 50 55 60 Pro Ser Thr Asp Asp
Arg Val Thr Trp Val Lys Ser Val Asp Glu Ala 65 70 75 80 Ile Ala Ala
Cys Gly Asp Val Pro Glu Ile Met Val Ile Gly Gly Gly 85 90 95 Arg
Val Tyr Glu Gln Phe Leu Pro Lys Ala Gln Lys Leu Tyr Leu Thr 100 105
110 His Ile Asp Ala Glu Val Glu Gly Asp Thr His Phe Pro Asp Tyr Glu
115 120 125 Pro Asp Asp Trp Glu Ser Val Phe Ser Glu Phe His Asp Ala
Asp Ala 130 135 140 Gln Asn Ser His Ser Tyr Cys Phe Glu Ile Leu Glu
Arg Arg 145 150 155 52245PRTArtificial
Sequencesource/note="Description of Artificial Sequence Synthetic
polypeptide" 52Ser Leu Ala Leu Ser Leu Thr Ala Asp Gln Met Val Ser
Ala Leu Leu 1 5 10 15 Asp Ala Glu Pro Pro Ile Leu Tyr Ser Glu Tyr
Asp Pro Thr Arg Pro 20 25 30 Phe Ser Glu Ala Ser Met Met Gly Leu
Leu Thr Asn Leu Ala Asp Arg 35 40 45 Glu Leu Val His Met Ile Asn
Trp Ala Lys Arg Val Pro Gly Phe Val 50 55 60 Asp Leu Ala Leu His
Asp Gln Val His Leu Leu Glu Cys Ala Trp Met 65 70 75 80 Glu Ile Leu
Met Ile Gly Leu Val Trp Arg Ser Met Glu His Pro Gly 85 90 95 Lys
Leu Leu Phe Ala Pro Asn Leu Leu Leu Asp Arg Asn Gln Gly Lys 100 105
110 Cys Val Glu Gly Gly Val Glu Ile Phe Asp Met Leu Leu Ala Thr Ser
115 120 125 Ser Arg Phe Arg Met Met Asn Leu Gln Gly Glu Glu Phe Val
Cys Leu 130 135 140 Lys Ser Ile Ile Leu Leu Asn Ser Gly Val Tyr Thr
Phe Leu Ser Ser 145 150 155 160 Thr Leu Lys Ser Leu Glu Glu Lys Asp
His Ile His Arg Val Leu Asp 165 170 175 Lys Ile Thr Asp Thr Leu Ile
His Leu Met Ala Lys Ala Gly Leu Thr 180 185 190 Leu Gln Gln Gln His
Gln Arg Leu Ala Gln Leu Leu Leu Ile Leu Ser 195 200 205 His Ile Arg
His Met Ser Ser Lys Arg Met Glu His Leu Tyr Ser Met 210 215 220 Lys
Cys Lys Asn Val Val Pro Leu Ser Asp Leu Leu Leu Glu Met Leu 225 230
235 240 Asp Ala His Arg Leu 245 5323PRTArtificial
Sequencesource/note="Description of Artificial Sequence Synthetic
peptide" 53Met Asp Tyr Lys Asp His Asp Gly Asp Tyr Lys Asp His Asp
Ile Asp 1 5 10 15 Tyr Lys Asp Asp Asp Asp Lys 20 5417PRTSimian
virus 40 54Met Ala Pro Lys Lys Lys Arg Lys Val Gly Ile His Gly Val
Pro Ala 1 5 10 15 Ala 556PRTArtificial
Sequencesource/note="Description of Artificial Sequence Synthetic
peptide" 55Gly Ser Gly Ser Gly Ser 1 5 561676PRTArtificial
Sequencesource/note="Description of Artificial Sequence Synthetic
polypeptide" 56Met Asp Tyr Lys Asp His Asp Gly Asp Tyr Lys Asp His
Asp Ile Asp 1 5 10 15 Tyr Lys Asp Asp Asp Asp Lys Met Ala Pro Lys
Lys Lys Arg Lys Val 20 25
30 Gly Ile His Gly Val Pro Ala Ala Gly Ser Ser Leu Ala Leu Ser Leu
35 40 45 Thr Ala Asp Gln Met Val Ser Ala Leu Leu Asp Ala Glu Pro
Pro Ile 50 55 60 Leu Tyr Ser Glu Tyr Asp Pro Thr Arg Pro Phe Ser
Glu Ala Ser Met 65 70 75 80 Met Gly Leu Leu Thr Asn Leu Ala Asp Arg
Glu Leu Val His Met Ile 85 90 95 Asn Trp Ala Lys Arg Val Pro Gly
Phe Val Asp Leu Ala Leu His Asp 100 105 110 Gln Val His Leu Leu Glu
Cys Ala Trp Met Glu Ile Leu Met Ile Gly 115 120 125 Leu Val Trp Arg
Ser Met Glu His Pro Gly Lys Leu Leu Phe Ala Pro 130 135 140 Asn Leu
Leu Leu Asp Arg Asn Gln Gly Lys Cys Val Glu Gly Gly Val 145 150 155
160 Glu Ile Phe Asp Met Leu Leu Ala Thr Ser Ser Arg Phe Arg Met Met
165 170 175 Asn Leu Gln Gly Glu Glu Phe Val Cys Leu Lys Ser Ile Ile
Leu Leu 180 185 190 Asn Ser Gly Val Tyr Thr Phe Leu Ser Ser Thr Leu
Lys Ser Leu Glu 195 200 205 Glu Lys Asp His Ile His Arg Val Leu Asp
Lys Ile Thr Asp Thr Leu 210 215 220 Ile His Leu Met Ala Lys Ala Gly
Leu Thr Leu Gln Gln Gln His Gln 225 230 235 240 Arg Leu Ala Gln Leu
Leu Leu Ile Leu Ser His Ile Arg His Met Ser 245 250 255 Ser Lys Arg
Met Glu His Leu Tyr Ser Met Lys Cys Lys Asn Val Val 260 265 270 Pro
Leu Ser Asp Leu Leu Leu Glu Met Leu Asp Ala His Arg Leu Gly 275 280
285 Ser Gly Ser Gly Ser Asp Lys Lys Tyr Ser Ile Gly Leu Asp Ile Gly
290 295 300 Thr Asn Ser Val Gly Trp Ala Val Ile Thr Asp Glu Tyr Lys
Val Pro 305 310 315 320 Ser Lys Lys Phe Lys Val Leu Gly Asn Thr Asp
Arg His Ser Ile Lys 325 330 335 Lys Asn Leu Ile Gly Ala Leu Leu Phe
Asp Ser Gly Glu Thr Ala Glu 340 345 350 Ala Thr Arg Leu Lys Arg Thr
Ala Arg Arg Arg Tyr Thr Arg Arg Lys 355 360 365 Asn Arg Ile Cys Tyr
Leu Gln Glu Ile Phe Ser Asn Glu Met Ala Lys 370 375 380 Val Asp Asp
Ser Phe Phe His Arg Leu Glu Glu Ser Phe Leu Val Glu 385 390 395 400
Glu Asp Lys Lys His Glu Arg His Pro Ile Phe Gly Asn Ile Val Asp 405
410 415 Glu Val Ala Tyr His Glu Lys Tyr Pro Thr Ile Tyr His Leu Arg
Lys 420 425 430 Lys Leu Val Asp Ser Thr Asp Lys Ala Asp Leu Arg Leu
Ile Tyr Leu 435 440 445 Ala Leu Ala His Met Ile Lys Phe Arg Gly His
Phe Leu Ile Glu Gly 450 455 460 Asp Leu Asn Pro Asp Asn Ser Asp Val
Asp Lys Leu Phe Ile Gln Leu 465 470 475 480 Val Gln Thr Tyr Asn Gln
Leu Phe Glu Glu Asn Pro Ile Asn Ala Ser 485 490 495 Gly Val Asp Ala
Lys Ala Ile Leu Ser Ala Arg Leu Ser Lys Ser Arg 500 505 510 Arg Leu
Glu Asn Leu Ile Ala Gln Leu Pro Gly Glu Lys Lys Asn Gly 515 520 525
Leu Phe Gly Asn Leu Ile Ala Leu Ser Leu Gly Leu Thr Pro Asn Phe 530
535 540 Lys Ser Asn Phe Asp Leu Ala Glu Asp Ala Lys Leu Gln Leu Ser
Lys 545 550 555 560 Asp Thr Tyr Asp Asp Asp Leu Asp Asn Leu Leu Ala
Gln Ile Gly Asp 565 570 575 Gln Tyr Ala Asp Leu Phe Leu Ala Ala Lys
Asn Leu Ser Asp Ala Ile 580 585 590 Leu Leu Ser Asp Ile Leu Arg Val
Asn Thr Glu Ile Thr Lys Ala Pro 595 600 605 Leu Ser Ala Ser Met Ile
Lys Arg Tyr Asp Glu His His Gln Asp Leu 610 615 620 Thr Leu Leu Lys
Ala Leu Val Arg Gln Gln Leu Pro Glu Lys Tyr Lys 625 630 635 640 Glu
Ile Phe Phe Asp Gln Ser Lys Asn Gly Tyr Ala Gly Tyr Ile Asp 645 650
655 Gly Gly Ala Ser Gln Glu Glu Phe Tyr Lys Phe Ile Lys Pro Ile Leu
660 665 670 Glu Lys Met Asp Gly Thr Glu Glu Leu Leu Val Lys Leu Asn
Arg Glu 675 680 685 Asp Leu Leu Arg Lys Gln Arg Thr Phe Asp Asn Gly
Ser Ile Pro His 690 695 700 Gln Ile His Leu Gly Glu Leu His Ala Ile
Leu Arg Arg Gln Glu Asp 705 710 715 720 Phe Tyr Pro Phe Leu Lys Asp
Asn Arg Glu Lys Ile Glu Lys Ile Leu 725 730 735 Thr Phe Arg Ile Pro
Tyr Tyr Val Gly Pro Leu Ala Arg Gly Asn Ser 740 745 750 Arg Phe Ala
Trp Met Thr Arg Lys Ser Glu Glu Thr Ile Thr Pro Trp 755 760 765 Asn
Phe Glu Glu Val Val Asp Lys Gly Ala Ser Ala Gln Ser Phe Ile 770 775
780 Glu Arg Met Thr Asn Phe Asp Lys Asn Leu Pro Asn Glu Lys Val Leu
785 790 795 800 Pro Lys His Ser Leu Leu Tyr Glu Tyr Phe Thr Val Tyr
Asn Glu Leu 805 810 815 Thr Lys Val Lys Tyr Val Thr Glu Gly Met Arg
Lys Pro Ala Phe Leu 820 825 830 Ser Gly Glu Gln Lys Lys Ala Ile Val
Asp Leu Leu Phe Lys Thr Asn 835 840 845 Arg Lys Val Thr Val Lys Gln
Leu Lys Glu Asp Tyr Phe Lys Lys Ile 850 855 860 Glu Cys Phe Asp Ser
Val Glu Ile Ser Gly Val Glu Asp Arg Phe Asn 865 870 875 880 Ala Ser
Leu Gly Thr Tyr His Asp Leu Leu Lys Ile Ile Lys Asp Lys 885 890 895
Asp Phe Leu Asp Asn Glu Glu Asn Glu Asp Ile Leu Glu Asp Ile Val 900
905 910 Leu Thr Leu Thr Leu Phe Glu Asp Arg Glu Met Ile Glu Glu Arg
Leu 915 920 925 Lys Thr Tyr Ala His Leu Phe Asp Asp Lys Val Met Lys
Gln Leu Lys 930 935 940 Arg Arg Arg Tyr Thr Gly Trp Gly Arg Leu Ser
Arg Lys Leu Ile Asn 945 950 955 960 Gly Ile Arg Asp Lys Gln Ser Gly
Lys Thr Ile Leu Asp Phe Leu Lys 965 970 975 Ser Asp Gly Phe Ala Asn
Arg Asn Phe Met Gln Leu Ile His Asp Asp 980 985 990 Ser Leu Thr Phe
Lys Glu Asp Ile Gln Lys Ala Gln Val Ser Gly Gln 995 1000 1005 Gly
Asp Ser Leu His Glu His Ile Ala Asn Leu Ala Gly Ser Pro 1010 1015
1020 Ala Ile Lys Lys Gly Ile Leu Gln Thr Val Lys Val Val Asp Glu
1025 1030 1035 Leu Val Lys Val Met Gly Arg His Lys Pro Glu Asn Ile
Val Ile 1040 1045 1050 Glu Met Ala Arg Glu Asn Gln Thr Thr Gln Lys
Gly Gln Lys Asn 1055 1060 1065 Ser Arg Glu Arg Met Lys Arg Ile Glu
Glu Gly Ile Lys Glu Leu 1070 1075 1080 Gly Ser Gln Ile Leu Lys Glu
His Pro Val Glu Asn Thr Gln Leu 1085 1090 1095 Gln Asn Glu Lys Leu
Tyr Leu Tyr Tyr Leu Gln Asn Gly Arg Asp 1100 1105 1110 Met Tyr Val
Asp Gln Glu Leu Asp Ile Asn Arg Leu Ser Asp Tyr 1115 1120 1125 Asp
Val Asp His Ile Val Pro Gln Ser Phe Leu Lys Asp Asp Ser 1130 1135
1140 Ile Asp Asn Lys Val Leu Thr Arg Ser Asp Lys Asn Arg Gly Lys
1145 1150 1155 Ser Asp Asn Val Pro Ser Glu Glu Val Val Lys Lys Met
Lys Asn 1160 1165 1170 Tyr Trp Arg Gln Leu Leu Asn Ala Lys Leu Ile
Thr Gln Arg Lys 1175 1180 1185 Phe Asp Asn Leu Thr Lys Ala Glu Arg
Gly Gly Leu Ser Glu Leu 1190 1195 1200 Asp Lys Ala Gly Phe Ile Lys
Arg Gln Leu Val Glu Thr Arg Gln 1205 1210 1215 Ile Thr Lys His Val
Ala Gln Ile Leu Asp Ser Arg Met Asn Thr 1220 1225 1230 Lys Tyr Asp
Glu Asn Asp Lys Leu Ile Arg Glu Val Lys Val Ile 1235 1240 1245 Thr
Leu Lys Ser Lys Leu Val Ser Asp Phe Arg Lys Asp Phe Gln 1250 1255
1260 Phe Tyr Lys Val Arg Glu Ile Asn Asn Tyr His His Ala His Asp
1265 1270 1275 Ala Tyr Leu Asn Ala Val Val Gly Thr Ala Leu Ile Lys
Lys Tyr 1280 1285 1290 Pro Lys Leu Glu Ser Glu Phe Val Tyr Gly Asp
Tyr Lys Val Tyr 1295 1300 1305 Asp Val Arg Lys Met Ile Ala Lys Ser
Glu Gln Glu Ile Gly Lys 1310 1315 1320 Ala Thr Ala Lys Tyr Phe Phe
Tyr Ser Asn Ile Met Asn Phe Phe 1325 1330 1335 Lys Thr Glu Ile Thr
Leu Ala Asn Gly Glu Ile Arg Lys Arg Pro 1340 1345 1350 Leu Ile Glu
Thr Asn Gly Glu Thr Gly Glu Ile Val Trp Asp Lys 1355 1360 1365 Gly
Arg Asp Phe Ala Thr Val Arg Lys Val Leu Ser Met Pro Gln 1370 1375
1380 Val Asn Ile Val Lys Lys Thr Glu Val Gln Thr Gly Gly Phe Ser
1385 1390 1395 Lys Glu Ser Ile Leu Pro Lys Arg Asn Ser Asp Lys Leu
Ile Ala 1400 1405 1410 Arg Lys Lys Asp Trp Asp Pro Lys Lys Tyr Gly
Gly Phe Asp Ser 1415 1420 1425 Pro Thr Val Ala Tyr Ser Val Leu Val
Val Ala Lys Val Glu Lys 1430 1435 1440 Gly Lys Ser Lys Lys Leu Lys
Ser Val Lys Glu Leu Leu Gly Ile 1445 1450 1455 Thr Ile Met Glu Arg
Ser Ser Phe Glu Lys Asn Pro Ile Asp Phe 1460 1465 1470 Leu Glu Ala
Lys Gly Tyr Lys Glu Val Lys Lys Asp Leu Ile Ile 1475 1480 1485 Lys
Leu Pro Lys Tyr Ser Leu Phe Glu Leu Glu Asn Gly Arg Lys 1490 1495
1500 Arg Met Leu Ala Ser Ala Gly Glu Leu Gln Lys Gly Asn Glu Leu
1505 1510 1515 Ala Leu Pro Ser Lys Tyr Val Asn Phe Leu Tyr Leu Ala
Ser His 1520 1525 1530 Tyr Glu Lys Leu Lys Gly Ser Pro Glu Asp Asn
Glu Gln Lys Gln 1535 1540 1545 Leu Phe Val Glu Gln His Lys His Tyr
Leu Asp Glu Ile Ile Glu 1550 1555 1560 Gln Ile Ser Glu Phe Ser Lys
Arg Val Ile Leu Ala Asp Ala Asn 1565 1570 1575 Leu Asp Lys Val Leu
Ser Ala Tyr Asn Lys His Arg Asp Lys Pro 1580 1585 1590 Ile Arg Glu
Gln Ala Glu Asn Ile Ile His Leu Phe Thr Leu Thr 1595 1600 1605 Asn
Leu Gly Ala Pro Ala Ala Phe Lys Tyr Phe Asp Thr Thr Ile 1610 1615
1620 Asp Arg Lys Arg Tyr Thr Ser Thr Lys Glu Val Leu Asp Ala Thr
1625 1630 1635 Leu Ile His Gln Ser Ile Thr Gly Leu Tyr Glu Thr Arg
Ile Asp 1640 1645 1650 Leu Ser Gln Leu Gly Gly Asp Lys Arg Pro Ala
Ala Thr Lys Lys 1655 1660 1665 Ala Gly Gln Ala Lys Lys Lys Lys 1670
1675 571668PRTArtificial Sequencesource/note="Description of
Artificial Sequence Synthetic polypeptide" 57Met Asp Tyr Lys Asp
His Asp Gly Asp Tyr Lys Asp His Asp Ile Asp 1 5 10 15 Tyr Lys Asp
Asp Asp Asp Lys Met Ala Pro Lys Lys Lys Arg Lys Val 20 25 30 Gly
Ile His Gly Val Pro Ala Ala Asp Lys Lys Tyr Ser Ile Gly Leu 35 40
45 Asp Ile Gly Thr Asn Ser Val Gly Trp Ala Val Ile Thr Asp Glu Tyr
50 55 60 Lys Val Pro Ser Lys Lys Phe Lys Val Leu Gly Asn Thr Asp
Arg His 65 70 75 80 Ser Ile Lys Lys Asn Leu Ile Gly Ala Leu Leu Phe
Asp Ser Gly Glu 85 90 95 Thr Ala Glu Ala Thr Arg Leu Lys Arg Thr
Ala Arg Arg Arg Tyr Thr 100 105 110 Arg Arg Lys Asn Arg Ile Cys Tyr
Leu Gln Glu Ile Phe Ser Asn Glu 115 120 125 Met Ala Lys Val Asp Asp
Ser Phe Phe His Arg Leu Glu Glu Ser Phe 130 135 140 Leu Val Glu Glu
Asp Lys Lys His Glu Arg His Pro Ile Phe Gly Asn 145 150 155 160 Ile
Val Asp Glu Val Ala Tyr His Glu Lys Tyr Pro Thr Ile Tyr His 165 170
175 Leu Arg Lys Lys Leu Val Asp Ser Thr Asp Lys Ala Asp Leu Arg Leu
180 185 190 Ile Tyr Leu Ala Leu Ala His Met Ile Lys Phe Arg Gly His
Phe Leu 195 200 205 Ile Glu Gly Asp Leu Asn Pro Asp Asn Ser Asp Val
Asp Lys Leu Phe 210 215 220 Ile Gln Leu Val Gln Thr Tyr Asn Gln Leu
Phe Glu Glu Asn Pro Ile 225 230 235 240 Asn Ala Ser Gly Val Asp Ala
Lys Ala Ile Leu Ser Ala Arg Leu Ser 245 250 255 Lys Ser Arg Arg Leu
Glu Asn Leu Ile Ala Gln Leu Pro Gly Glu Lys 260 265 270 Lys Asn Gly
Leu Phe Gly Asn Leu Ile Ala Leu Ser Leu Gly Leu Thr 275 280 285 Pro
Asn Phe Lys Ser Asn Phe Asp Leu Ala Glu Asp Ala Lys Leu Gln 290 295
300 Leu Ser Lys Asp Thr Tyr Asp Asp Asp Leu Asp Asn Leu Leu Ala Gln
305 310 315 320 Ile Gly Asp Gln Tyr Ala Asp Leu Phe Leu Ala Ala Lys
Asn Leu Ser 325 330 335 Asp Ala Ile Leu Leu Ser Asp Ile Leu Arg Val
Asn Thr Glu Ile Thr 340 345 350 Lys Ala Pro Leu Ser Ala Ser Met Ile
Lys Arg Tyr Asp Glu His His 355 360 365 Gln Asp Leu Thr Leu Leu Lys
Ala Leu Val Arg Gln Gln Leu Pro Glu 370 375 380 Lys Tyr Lys Glu Ile
Phe Phe Asp Gln Ser Lys Asn Gly Tyr Ala Gly 385 390 395 400 Tyr Ile
Asp Gly Gly Ala Ser Gln Glu Glu Phe Tyr Lys Phe Ile Lys 405 410 415
Pro Ile Leu Glu Lys Met Asp Gly Thr Glu Glu Leu Leu Val Lys Leu 420
425 430 Asn Arg Glu Asp Leu Leu Arg Lys Gln Arg Thr Phe Asp Asn Gly
Ser 435 440 445 Ile Pro His Gln Ile His Leu Gly Glu Leu His Ala Ile
Leu Arg Arg 450 455 460 Gln Glu Asp Phe Tyr Pro Phe Leu Lys Asp Asn
Arg Glu Lys Ile Glu 465 470 475 480 Lys Ile Leu Thr Phe Arg Ile Pro
Tyr Tyr Val Gly Pro Leu Ala Arg 485 490 495 Gly Asn Ser Arg Phe Ala
Trp Met Thr Arg Lys Ser Glu Glu Thr Ile 500 505 510 Thr Pro Trp Asn
Phe Glu Glu Val Val Asp Lys Gly Ala Ser Ala Gln 515 520 525 Ser Phe
Ile Glu Arg Met Thr Asn Phe Asp Lys Asn Leu Pro Asn Glu 530 535 540
Lys Val Leu Pro Lys His Ser Leu Leu Tyr Glu Tyr Phe Thr Val Tyr 545
550 555 560 Asn Glu Leu Thr Lys Val Lys Tyr Val Thr Glu Gly Met Arg
Lys Pro 565 570 575 Ala Phe Leu Ser Gly Glu Gln Lys Lys Ala Ile Val
Asp Leu Leu Phe 580 585 590 Lys Thr Asn Arg Lys Val Thr Val Lys Gln
Leu Lys Glu Asp Tyr Phe 595 600 605 Lys Lys Ile Glu Cys Phe
Asp Ser Val Glu Ile Ser Gly Val Glu Asp 610 615 620 Arg Phe Asn Ala
Ser Leu Gly Thr Tyr His Asp Leu Leu Lys Ile Ile 625 630 635 640 Lys
Asp Lys Asp Phe Leu Asp Asn Glu Glu Asn Glu Asp Ile Leu Glu 645 650
655 Asp Ile Val Leu Thr Leu Thr Leu Phe Glu Asp Arg Glu Met Ile Glu
660 665 670 Glu Arg Leu Lys Thr Tyr Ala His Leu Phe Asp Asp Lys Val
Met Lys 675 680 685 Gln Leu Lys Arg Arg Arg Tyr Thr Gly Trp Gly Arg
Leu Ser Arg Lys 690 695 700 Leu Ile Asn Gly Ile Arg Asp Lys Gln Ser
Gly Lys Thr Ile Leu Asp 705 710 715 720 Phe Leu Lys Ser Asp Gly Phe
Ala Asn Arg Asn Phe Met Gln Leu Ile 725 730 735 His Asp Asp Ser Leu
Thr Phe Lys Glu Asp Ile Gln Lys Ala Gln Val 740 745 750 Ser Gly Gln
Gly Asp Ser Leu His Glu His Ile Ala Asn Leu Ala Gly 755 760 765 Ser
Pro Ala Ile Lys Lys Gly Ile Leu Gln Thr Val Lys Val Val Asp 770 775
780 Glu Leu Val Lys Val Met Gly Arg His Lys Pro Glu Asn Ile Val Ile
785 790 795 800 Glu Met Ala Arg Glu Asn Gln Thr Thr Gln Lys Gly Gln
Lys Asn Ser 805 810 815 Arg Glu Arg Met Lys Arg Ile Glu Glu Gly Ile
Lys Glu Leu Gly Ser 820 825 830 Gln Ile Leu Lys Glu His Pro Val Glu
Asn Thr Gln Leu Gln Asn Glu 835 840 845 Lys Leu Tyr Leu Tyr Tyr Leu
Gln Asn Gly Arg Asp Met Tyr Val Asp 850 855 860 Gln Glu Leu Asp Ile
Asn Arg Leu Ser Asp Tyr Asp Val Asp His Ile 865 870 875 880 Val Pro
Gln Ser Phe Leu Lys Asp Asp Ser Ile Asp Asn Lys Val Leu 885 890 895
Thr Arg Ser Asp Lys Asn Arg Gly Lys Ser Asp Asn Val Pro Ser Glu 900
905 910 Glu Val Val Lys Lys Met Lys Asn Tyr Trp Arg Gln Leu Leu Asn
Ala 915 920 925 Lys Leu Ile Thr Gln Arg Lys Phe Asp Asn Leu Thr Lys
Ala Glu Arg 930 935 940 Gly Gly Leu Ser Glu Leu Asp Lys Ala Gly Phe
Ile Lys Arg Gln Leu 945 950 955 960 Val Glu Thr Arg Gln Ile Thr Lys
His Val Ala Gln Ile Leu Asp Ser 965 970 975 Arg Met Asn Thr Lys Tyr
Asp Glu Asn Asp Lys Leu Ile Arg Glu Val 980 985 990 Lys Val Ile Thr
Leu Lys Ser Lys Leu Val Ser Asp Phe Arg Lys Asp 995 1000 1005 Phe
Gln Phe Tyr Lys Val Arg Glu Ile Asn Asn Tyr His His Ala 1010 1015
1020 His Asp Ala Tyr Leu Asn Ala Val Val Gly Thr Ala Leu Ile Lys
1025 1030 1035 Lys Tyr Pro Lys Leu Glu Ser Glu Phe Val Tyr Gly Asp
Tyr Lys 1040 1045 1050 Val Tyr Asp Val Arg Lys Met Ile Ala Lys Ser
Glu Gln Glu Ile 1055 1060 1065 Gly Lys Ala Thr Ala Lys Tyr Phe Phe
Tyr Ser Asn Ile Met Asn 1070 1075 1080 Phe Phe Lys Thr Glu Ile Thr
Leu Ala Asn Gly Glu Ile Arg Lys 1085 1090 1095 Arg Pro Leu Ile Glu
Thr Asn Gly Glu Thr Gly Glu Ile Val Trp 1100 1105 1110 Asp Lys Gly
Arg Asp Phe Ala Thr Val Arg Lys Val Leu Ser Met 1115 1120 1125 Pro
Gln Val Asn Ile Val Lys Lys Thr Glu Val Gln Thr Gly Gly 1130 1135
1140 Phe Ser Lys Glu Ser Ile Leu Pro Lys Arg Asn Ser Asp Lys Leu
1145 1150 1155 Ile Ala Arg Lys Lys Asp Trp Asp Pro Lys Lys Tyr Gly
Gly Phe 1160 1165 1170 Asp Ser Pro Thr Val Ala Tyr Ser Val Leu Val
Val Ala Lys Val 1175 1180 1185 Glu Lys Gly Lys Ser Lys Lys Leu Lys
Ser Val Lys Glu Leu Leu 1190 1195 1200 Gly Ile Thr Ile Met Glu Arg
Ser Ser Phe Glu Lys Asn Pro Ile 1205 1210 1215 Asp Phe Leu Glu Ala
Lys Gly Tyr Lys Glu Val Lys Lys Asp Leu 1220 1225 1230 Ile Ile Lys
Leu Pro Lys Tyr Ser Leu Phe Glu Leu Glu Asn Gly 1235 1240 1245 Arg
Lys Arg Met Leu Ala Ser Ala Gly Glu Leu Gln Lys Gly Asn 1250 1255
1260 Glu Leu Ala Leu Pro Ser Lys Tyr Val Asn Phe Leu Tyr Leu Ala
1265 1270 1275 Ser His Tyr Glu Lys Leu Lys Gly Ser Pro Glu Asp Asn
Glu Gln 1280 1285 1290 Lys Gln Leu Phe Val Glu Gln His Lys His Tyr
Leu Asp Glu Ile 1295 1300 1305 Ile Glu Gln Ile Ser Glu Phe Ser Lys
Arg Val Ile Leu Ala Asp 1310 1315 1320 Ala Asn Leu Asp Lys Val Leu
Ser Ala Tyr Asn Lys His Arg Asp 1325 1330 1335 Lys Pro Ile Arg Glu
Gln Ala Glu Asn Ile Ile His Leu Phe Thr 1340 1345 1350 Leu Thr Asn
Leu Gly Ala Pro Ala Ala Phe Lys Tyr Phe Asp Thr 1355 1360 1365 Thr
Ile Asp Arg Lys Arg Tyr Thr Ser Thr Lys Glu Val Leu Asp 1370 1375
1380 Ala Thr Leu Ile His Gln Ser Ile Thr Gly Leu Tyr Glu Thr Arg
1385 1390 1395 Ile Asp Leu Ser Gln Leu Gly Gly Asp Lys Arg Pro Ala
Ala Thr 1400 1405 1410 Lys Lys Ala Gly Gln Ala Lys Lys Lys Lys Ser
Leu Ala Leu Ser 1415 1420 1425 Leu Thr Ala Asp Gln Met Val Ser Ala
Leu Leu Asp Ala Glu Pro 1430 1435 1440 Pro Ile Leu Tyr Ser Glu Tyr
Asp Pro Thr Arg Pro Phe Ser Glu 1445 1450 1455 Ala Ser Met Met Gly
Leu Leu Thr Asn Leu Ala Asp Arg Glu Leu 1460 1465 1470 Val His Met
Ile Asn Trp Ala Lys Arg Val Pro Gly Phe Val Asp 1475 1480 1485 Leu
Ala Leu His Asp Gln Val His Leu Leu Glu Cys Ala Trp Met 1490 1495
1500 Glu Ile Leu Met Ile Gly Leu Val Trp Arg Ser Met Glu His Pro
1505 1510 1515 Gly Lys Leu Leu Phe Ala Pro Asn Leu Leu Leu Asp Arg
Asn Gln 1520 1525 1530 Gly Lys Cys Val Glu Gly Gly Val Glu Ile Phe
Asp Met Leu Leu 1535 1540 1545 Ala Thr Ser Ser Arg Phe Arg Met Met
Asn Leu Gln Gly Glu Glu 1550 1555 1560 Phe Val Cys Leu Lys Ser Ile
Ile Leu Leu Asn Ser Gly Val Tyr 1565 1570 1575 Thr Phe Leu Ser Ser
Thr Leu Lys Ser Leu Glu Glu Lys Asp His 1580 1585 1590 Ile His Arg
Val Leu Asp Lys Ile Thr Asp Thr Leu Ile His Leu 1595 1600 1605 Met
Ala Lys Ala Gly Leu Thr Leu Gln Gln Gln His Gln Arg Leu 1610 1615
1620 Ala Gln Leu Leu Leu Ile Leu Ser His Ile Arg His Met Ser Ser
1625 1630 1635 Lys Arg Met Glu His Leu Tyr Ser Met Lys Cys Lys Asn
Val Val 1640 1645 1650 Pro Leu Ser Asp Leu Leu Leu Glu Met Leu Asp
Ala His Arg Leu 1655 1660 1665 581921PRTArtificial
Sequencesource/note="Description of Artificial Sequence Synthetic
polypeptide" 58Met Asp Tyr Lys Asp His Asp Gly Asp Tyr Lys Asp His
Asp Ile Asp 1 5 10 15 Tyr Lys Asp Asp Asp Asp Lys Met Ala Pro Lys
Lys Lys Arg Lys Val 20 25 30 Gly Ile His Gly Val Pro Ala Ala Gly
Ser Ser Leu Ala Leu Ser Leu 35 40 45 Thr Ala Asp Gln Met Val Ser
Ala Leu Leu Asp Ala Glu Pro Pro Ile 50 55 60 Leu Tyr Ser Glu Tyr
Asp Pro Thr Arg Pro Phe Ser Glu Ala Ser Met 65 70 75 80 Met Gly Leu
Leu Thr Asn Leu Ala Asp Arg Glu Leu Val His Met Ile 85 90 95 Asn
Trp Ala Lys Arg Val Pro Gly Phe Val Asp Leu Ala Leu His Asp 100 105
110 Gln Val His Leu Leu Glu Cys Ala Trp Met Glu Ile Leu Met Ile Gly
115 120 125 Leu Val Trp Arg Ser Met Glu His Pro Gly Lys Leu Leu Phe
Ala Pro 130 135 140 Asn Leu Leu Leu Asp Arg Asn Gln Gly Lys Cys Val
Glu Gly Gly Val 145 150 155 160 Glu Ile Phe Asp Met Leu Leu Ala Thr
Ser Ser Arg Phe Arg Met Met 165 170 175 Asn Leu Gln Gly Glu Glu Phe
Val Cys Leu Lys Ser Ile Ile Leu Leu 180 185 190 Asn Ser Gly Val Tyr
Thr Phe Leu Ser Ser Thr Leu Lys Ser Leu Glu 195 200 205 Glu Lys Asp
His Ile His Arg Val Leu Asp Lys Ile Thr Asp Thr Leu 210 215 220 Ile
His Leu Met Ala Lys Ala Gly Leu Thr Leu Gln Gln Gln His Gln 225 230
235 240 Arg Leu Ala Gln Leu Leu Leu Ile Leu Ser His Ile Arg His Met
Ser 245 250 255 Ser Lys Arg Met Glu His Leu Tyr Ser Met Lys Cys Lys
Asn Val Val 260 265 270 Pro Leu Ser Asp Leu Leu Leu Glu Met Leu Asp
Ala His Arg Leu Gly 275 280 285 Ser Gly Ser Gly Ser Asp Lys Lys Tyr
Ser Ile Gly Leu Asp Ile Gly 290 295 300 Thr Asn Ser Val Gly Trp Ala
Val Ile Thr Asp Glu Tyr Lys Val Pro 305 310 315 320 Ser Lys Lys Phe
Lys Val Leu Gly Asn Thr Asp Arg His Ser Ile Lys 325 330 335 Lys Asn
Leu Ile Gly Ala Leu Leu Phe Asp Ser Gly Glu Thr Ala Glu 340 345 350
Ala Thr Arg Leu Lys Arg Thr Ala Arg Arg Arg Tyr Thr Arg Arg Lys 355
360 365 Asn Arg Ile Cys Tyr Leu Gln Glu Ile Phe Ser Asn Glu Met Ala
Lys 370 375 380 Val Asp Asp Ser Phe Phe His Arg Leu Glu Glu Ser Phe
Leu Val Glu 385 390 395 400 Glu Asp Lys Lys His Glu Arg His Pro Ile
Phe Gly Asn Ile Val Asp 405 410 415 Glu Val Ala Tyr His Glu Lys Tyr
Pro Thr Ile Tyr His Leu Arg Lys 420 425 430 Lys Leu Val Asp Ser Thr
Asp Lys Ala Asp Leu Arg Leu Ile Tyr Leu 435 440 445 Ala Leu Ala His
Met Ile Lys Phe Arg Gly His Phe Leu Ile Glu Gly 450 455 460 Asp Leu
Asn Pro Asp Asn Ser Asp Val Asp Lys Leu Phe Ile Gln Leu 465 470 475
480 Val Gln Thr Tyr Asn Gln Leu Phe Glu Glu Asn Pro Ile Asn Ala Ser
485 490 495 Gly Val Asp Ala Lys Ala Ile Leu Ser Ala Arg Leu Ser Lys
Ser Arg 500 505 510 Arg Leu Glu Asn Leu Ile Ala Gln Leu Pro Gly Glu
Lys Lys Asn Gly 515 520 525 Leu Phe Gly Asn Leu Ile Ala Leu Ser Leu
Gly Leu Thr Pro Asn Phe 530 535 540 Lys Ser Asn Phe Asp Leu Ala Glu
Asp Ala Lys Leu Gln Leu Ser Lys 545 550 555 560 Asp Thr Tyr Asp Asp
Asp Leu Asp Asn Leu Leu Ala Gln Ile Gly Asp 565 570 575 Gln Tyr Ala
Asp Leu Phe Leu Ala Ala Lys Asn Leu Ser Asp Ala Ile 580 585 590 Leu
Leu Ser Asp Ile Leu Arg Val Asn Thr Glu Ile Thr Lys Ala Pro 595 600
605 Leu Ser Ala Ser Met Ile Lys Arg Tyr Asp Glu His His Gln Asp Leu
610 615 620 Thr Leu Leu Lys Ala Leu Val Arg Gln Gln Leu Pro Glu Lys
Tyr Lys 625 630 635 640 Glu Ile Phe Phe Asp Gln Ser Lys Asn Gly Tyr
Ala Gly Tyr Ile Asp 645 650 655 Gly Gly Ala Ser Gln Glu Glu Phe Tyr
Lys Phe Ile Lys Pro Ile Leu 660 665 670 Glu Lys Met Asp Gly Thr Glu
Glu Leu Leu Val Lys Leu Asn Arg Glu 675 680 685 Asp Leu Leu Arg Lys
Gln Arg Thr Phe Asp Asn Gly Ser Ile Pro His 690 695 700 Gln Ile His
Leu Gly Glu Leu His Ala Ile Leu Arg Arg Gln Glu Asp 705 710 715 720
Phe Tyr Pro Phe Leu Lys Asp Asn Arg Glu Lys Ile Glu Lys Ile Leu 725
730 735 Thr Phe Arg Ile Pro Tyr Tyr Val Gly Pro Leu Ala Arg Gly Asn
Ser 740 745 750 Arg Phe Ala Trp Met Thr Arg Lys Ser Glu Glu Thr Ile
Thr Pro Trp 755 760 765 Asn Phe Glu Glu Val Val Asp Lys Gly Ala Ser
Ala Gln Ser Phe Ile 770 775 780 Glu Arg Met Thr Asn Phe Asp Lys Asn
Leu Pro Asn Glu Lys Val Leu 785 790 795 800 Pro Lys His Ser Leu Leu
Tyr Glu Tyr Phe Thr Val Tyr Asn Glu Leu 805 810 815 Thr Lys Val Lys
Tyr Val Thr Glu Gly Met Arg Lys Pro Ala Phe Leu 820 825 830 Ser Gly
Glu Gln Lys Lys Ala Ile Val Asp Leu Leu Phe Lys Thr Asn 835 840 845
Arg Lys Val Thr Val Lys Gln Leu Lys Glu Asp Tyr Phe Lys Lys Ile 850
855 860 Glu Cys Phe Asp Ser Val Glu Ile Ser Gly Val Glu Asp Arg Phe
Asn 865 870 875 880 Ala Ser Leu Gly Thr Tyr His Asp Leu Leu Lys Ile
Ile Lys Asp Lys 885 890 895 Asp Phe Leu Asp Asn Glu Glu Asn Glu Asp
Ile Leu Glu Asp Ile Val 900 905 910 Leu Thr Leu Thr Leu Phe Glu Asp
Arg Glu Met Ile Glu Glu Arg Leu 915 920 925 Lys Thr Tyr Ala His Leu
Phe Asp Asp Lys Val Met Lys Gln Leu Lys 930 935 940 Arg Arg Arg Tyr
Thr Gly Trp Gly Arg Leu Ser Arg Lys Leu Ile Asn 945 950 955 960 Gly
Ile Arg Asp Lys Gln Ser Gly Lys Thr Ile Leu Asp Phe Leu Lys 965 970
975 Ser Asp Gly Phe Ala Asn Arg Asn Phe Met Gln Leu Ile His Asp Asp
980 985 990 Ser Leu Thr Phe Lys Glu Asp Ile Gln Lys Ala Gln Val Ser
Gly Gln 995 1000 1005 Gly Asp Ser Leu His Glu His Ile Ala Asn Leu
Ala Gly Ser Pro 1010 1015 1020 Ala Ile Lys Lys Gly Ile Leu Gln Thr
Val Lys Val Val Asp Glu 1025 1030 1035 Leu Val Lys Val Met Gly Arg
His Lys Pro Glu Asn Ile Val Ile 1040 1045 1050 Glu Met Ala Arg Glu
Asn Gln Thr Thr Gln Lys Gly Gln Lys Asn 1055 1060 1065 Ser Arg Glu
Arg Met Lys Arg Ile Glu Glu Gly Ile Lys Glu Leu 1070 1075 1080 Gly
Ser Gln Ile Leu Lys Glu His Pro Val Glu Asn Thr Gln Leu 1085 1090
1095 Gln Asn Glu Lys Leu Tyr Leu Tyr Tyr Leu Gln Asn Gly Arg Asp
1100 1105 1110 Met Tyr Val Asp Gln Glu Leu Asp Ile Asn Arg Leu Ser
Asp Tyr 1115 1120 1125 Asp Val Asp His Ile Val Pro Gln Ser Phe Leu
Lys Asp Asp Ser 1130 1135 1140 Ile Asp Asn Lys Val Leu Thr Arg Ser
Asp Lys Asn Arg Gly Lys 1145 1150 1155 Ser Asp Asn Val Pro Ser Glu
Glu Val Val Lys Lys Met Lys Asn 1160 1165 1170 Tyr Trp Arg Gln Leu
Leu Asn Ala Lys Leu Ile Thr Gln Arg Lys 1175 1180 1185
Phe Asp Asn Leu Thr Lys Ala Glu Arg Gly Gly Leu Ser Glu Leu 1190
1195 1200 Asp Lys Ala Gly Phe Ile Lys Arg Gln Leu Val Glu Thr Arg
Gln 1205 1210 1215 Ile Thr Lys His Val Ala Gln Ile Leu Asp Ser Arg
Met Asn Thr 1220 1225 1230 Lys Tyr Asp Glu Asn Asp Lys Leu Ile Arg
Glu Val Lys Val Ile 1235 1240 1245 Thr Leu Lys Ser Lys Leu Val Ser
Asp Phe Arg Lys Asp Phe Gln 1250 1255 1260 Phe Tyr Lys Val Arg Glu
Ile Asn Asn Tyr His His Ala His Asp 1265 1270 1275 Ala Tyr Leu Asn
Ala Val Val Gly Thr Ala Leu Ile Lys Lys Tyr 1280 1285 1290 Pro Lys
Leu Glu Ser Glu Phe Val Tyr Gly Asp Tyr Lys Val Tyr 1295 1300 1305
Asp Val Arg Lys Met Ile Ala Lys Ser Glu Gln Glu Ile Gly Lys 1310
1315 1320 Ala Thr Ala Lys Tyr Phe Phe Tyr Ser Asn Ile Met Asn Phe
Phe 1325 1330 1335 Lys Thr Glu Ile Thr Leu Ala Asn Gly Glu Ile Arg
Lys Arg Pro 1340 1345 1350 Leu Ile Glu Thr Asn Gly Glu Thr Gly Glu
Ile Val Trp Asp Lys 1355 1360 1365 Gly Arg Asp Phe Ala Thr Val Arg
Lys Val Leu Ser Met Pro Gln 1370 1375 1380 Val Asn Ile Val Lys Lys
Thr Glu Val Gln Thr Gly Gly Phe Ser 1385 1390 1395 Lys Glu Ser Ile
Leu Pro Lys Arg Asn Ser Asp Lys Leu Ile Ala 1400 1405 1410 Arg Lys
Lys Asp Trp Asp Pro Lys Lys Tyr Gly Gly Phe Asp Ser 1415 1420 1425
Pro Thr Val Ala Tyr Ser Val Leu Val Val Ala Lys Val Glu Lys 1430
1435 1440 Gly Lys Ser Lys Lys Leu Lys Ser Val Lys Glu Leu Leu Gly
Ile 1445 1450 1455 Thr Ile Met Glu Arg Ser Ser Phe Glu Lys Asn Pro
Ile Asp Phe 1460 1465 1470 Leu Glu Ala Lys Gly Tyr Lys Glu Val Lys
Lys Asp Leu Ile Ile 1475 1480 1485 Lys Leu Pro Lys Tyr Ser Leu Phe
Glu Leu Glu Asn Gly Arg Lys 1490 1495 1500 Arg Met Leu Ala Ser Ala
Gly Glu Leu Gln Lys Gly Asn Glu Leu 1505 1510 1515 Ala Leu Pro Ser
Lys Tyr Val Asn Phe Leu Tyr Leu Ala Ser His 1520 1525 1530 Tyr Glu
Lys Leu Lys Gly Ser Pro Glu Asp Asn Glu Gln Lys Gln 1535 1540 1545
Leu Phe Val Glu Gln His Lys His Tyr Leu Asp Glu Ile Ile Glu 1550
1555 1560 Gln Ile Ser Glu Phe Ser Lys Arg Val Ile Leu Ala Asp Ala
Asn 1565 1570 1575 Leu Asp Lys Val Leu Ser Ala Tyr Asn Lys His Arg
Asp Lys Pro 1580 1585 1590 Ile Arg Glu Gln Ala Glu Asn Ile Ile His
Leu Phe Thr Leu Thr 1595 1600 1605 Asn Leu Gly Ala Pro Ala Ala Phe
Lys Tyr Phe Asp Thr Thr Ile 1610 1615 1620 Asp Arg Lys Arg Tyr Thr
Ser Thr Lys Glu Val Leu Asp Ala Thr 1625 1630 1635 Leu Ile His Gln
Ser Ile Thr Gly Leu Tyr Glu Thr Arg Ile Asp 1640 1645 1650 Leu Ser
Gln Leu Gly Gly Asp Lys Arg Pro Ala Ala Thr Lys Lys 1655 1660 1665
Ala Gly Gln Ala Lys Lys Lys Lys Ser Leu Ala Leu Ser Leu Thr 1670
1675 1680 Ala Asp Gln Met Val Ser Ala Leu Leu Asp Ala Glu Pro Pro
Ile 1685 1690 1695 Leu Tyr Ser Glu Tyr Asp Pro Thr Arg Pro Phe Ser
Glu Ala Ser 1700 1705 1710 Met Met Gly Leu Leu Thr Asn Leu Ala Asp
Arg Glu Leu Val His 1715 1720 1725 Met Ile Asn Trp Ala Lys Arg Val
Pro Gly Phe Val Asp Leu Ala 1730 1735 1740 Leu His Asp Gln Val His
Leu Leu Glu Cys Ala Trp Met Glu Ile 1745 1750 1755 Leu Met Ile Gly
Leu Val Trp Arg Ser Met Glu His Pro Gly Lys 1760 1765 1770 Leu Leu
Phe Ala Pro Asn Leu Leu Leu Asp Arg Asn Gln Gly Lys 1775 1780 1785
Cys Val Glu Gly Gly Val Glu Ile Phe Asp Met Leu Leu Ala Thr 1790
1795 1800 Ser Ser Arg Phe Arg Met Met Asn Leu Gln Gly Glu Glu Phe
Val 1805 1810 1815 Cys Leu Lys Ser Ile Ile Leu Leu Asn Ser Gly Val
Tyr Thr Phe 1820 1825 1830 Leu Ser Ser Thr Leu Lys Ser Leu Glu Glu
Lys Asp His Ile His 1835 1840 1845 Arg Val Leu Asp Lys Ile Thr Asp
Thr Leu Ile His Leu Met Ala 1850 1855 1860 Lys Ala Gly Leu Thr Leu
Gln Gln Gln His Gln Arg Leu Ala Gln 1865 1870 1875 Leu Leu Leu Ile
Leu Ser His Ile Arg His Met Ser Ser Lys Arg 1880 1885 1890 Met Glu
His Leu Tyr Ser Met Lys Cys Lys Asn Val Val Pro Leu 1895 1900 1905
Ser Asp Leu Leu Leu Glu Met Leu Asp Ala His Arg Leu 1910 1915 1920
591589PRTArtificial Sequencesource/note="Description of Artificial
Sequence Synthetic polypeptide" 59Met Asp Tyr Lys Asp His Asp Gly
Asp Tyr Lys Asp His Asp Ile Asp 1 5 10 15 Tyr Lys Asp Asp Asp Asp
Lys Met Ala Pro Lys Lys Lys Arg Lys Val 20 25 30 Gly Ile His Gly
Val Pro Ala Ala Gly Ser Ile Ser Leu Ile Ala Ala 35 40 45 Leu Ala
Val Asp Tyr Val Ile Gly Met Glu Asn Ala Met Pro Trp Asn 50 55 60
Leu Pro Ala Asp Leu Ala Trp Phe Lys Arg Asn Thr Leu Asn Lys Pro 65
70 75 80 Val Ile Met Gly Arg His Thr Trp Glu Ser Ile Gly Arg Pro
Leu Pro 85 90 95 Gly Arg Lys Asn Ile Ile Leu Ser Ser Gln Pro Ser
Thr Asp Asp Arg 100 105 110 Val Thr Trp Val Lys Ser Val Asp Glu Ala
Ile Ala Ala Cys Gly Asp 115 120 125 Val Pro Glu Ile Met Val Ile Gly
Gly Gly Arg Val Ile Glu Gln Phe 130 135 140 Leu Pro Lys Ala Gln Lys
Leu Tyr Leu Thr His Ile Asp Ala Glu Val 145 150 155 160 Glu Gly Asp
Thr His Phe Pro Asp Tyr Glu Pro Asp Asp Trp Glu Ser 165 170 175 Val
Phe Ser Glu Phe His Asp Ala Asp Ala Gln Asn Ser His Ser Tyr 180 185
190 Cys Phe Glu Ile Leu Glu Arg Arg Gly Ser Gly Ser Gly Ser Asp Lys
195 200 205 Lys Tyr Ser Ile Gly Leu Asp Ile Gly Thr Asn Ser Val Gly
Trp Ala 210 215 220 Val Ile Thr Asp Glu Tyr Lys Val Pro Ser Lys Lys
Phe Lys Val Leu 225 230 235 240 Gly Asn Thr Asp Arg His Ser Ile Lys
Lys Asn Leu Ile Gly Ala Leu 245 250 255 Leu Phe Asp Ser Gly Glu Thr
Ala Glu Ala Thr Arg Leu Lys Arg Thr 260 265 270 Ala Arg Arg Arg Tyr
Thr Arg Arg Lys Asn Arg Ile Cys Tyr Leu Gln 275 280 285 Glu Ile Phe
Ser Asn Glu Met Ala Lys Val Asp Asp Ser Phe Phe His 290 295 300 Arg
Leu Glu Glu Ser Phe Leu Val Glu Glu Asp Lys Lys His Glu Arg 305 310
315 320 His Pro Ile Phe Gly Asn Ile Val Asp Glu Val Ala Tyr His Glu
Lys 325 330 335 Tyr Pro Thr Ile Tyr His Leu Arg Lys Lys Leu Val Asp
Ser Thr Asp 340 345 350 Lys Ala Asp Leu Arg Leu Ile Tyr Leu Ala Leu
Ala His Met Ile Lys 355 360 365 Phe Arg Gly His Phe Leu Ile Glu Gly
Asp Leu Asn Pro Asp Asn Ser 370 375 380 Asp Val Asp Lys Leu Phe Ile
Gln Leu Val Gln Thr Tyr Asn Gln Leu 385 390 395 400 Phe Glu Glu Asn
Pro Ile Asn Ala Ser Gly Val Asp Ala Lys Ala Ile 405 410 415 Leu Ser
Ala Arg Leu Ser Lys Ser Arg Arg Leu Glu Asn Leu Ile Ala 420 425 430
Gln Leu Pro Gly Glu Lys Lys Asn Gly Leu Phe Gly Asn Leu Ile Ala 435
440 445 Leu Ser Leu Gly Leu Thr Pro Asn Phe Lys Ser Asn Phe Asp Leu
Ala 450 455 460 Glu Asp Ala Lys Leu Gln Leu Ser Lys Asp Thr Tyr Asp
Asp Asp Leu 465 470 475 480 Asp Asn Leu Leu Ala Gln Ile Gly Asp Gln
Tyr Ala Asp Leu Phe Leu 485 490 495 Ala Ala Lys Asn Leu Ser Asp Ala
Ile Leu Leu Ser Asp Ile Leu Arg 500 505 510 Val Asn Thr Glu Ile Thr
Lys Ala Pro Leu Ser Ala Ser Met Ile Lys 515 520 525 Arg Tyr Asp Glu
His His Gln Asp Leu Thr Leu Leu Lys Ala Leu Val 530 535 540 Arg Gln
Gln Leu Pro Glu Lys Tyr Lys Glu Ile Phe Phe Asp Gln Ser 545 550 555
560 Lys Asn Gly Tyr Ala Gly Tyr Ile Asp Gly Gly Ala Ser Gln Glu Glu
565 570 575 Phe Tyr Lys Phe Ile Lys Pro Ile Leu Glu Lys Met Asp Gly
Thr Glu 580 585 590 Glu Leu Leu Val Lys Leu Asn Arg Glu Asp Leu Leu
Arg Lys Gln Arg 595 600 605 Thr Phe Asp Asn Gly Ser Ile Pro His Gln
Ile His Leu Gly Glu Leu 610 615 620 His Ala Ile Leu Arg Arg Gln Glu
Asp Phe Tyr Pro Phe Leu Lys Asp 625 630 635 640 Asn Arg Glu Lys Ile
Glu Lys Ile Leu Thr Phe Arg Ile Pro Tyr Tyr 645 650 655 Val Gly Pro
Leu Ala Arg Gly Asn Ser Arg Phe Ala Trp Met Thr Arg 660 665 670 Lys
Ser Glu Glu Thr Ile Thr Pro Trp Asn Phe Glu Glu Val Val Asp 675 680
685 Lys Gly Ala Ser Ala Gln Ser Phe Ile Glu Arg Met Thr Asn Phe Asp
690 695 700 Lys Asn Leu Pro Asn Glu Lys Val Leu Pro Lys His Ser Leu
Leu Tyr 705 710 715 720 Glu Tyr Phe Thr Val Tyr Asn Glu Leu Thr Lys
Val Lys Tyr Val Thr 725 730 735 Glu Gly Met Arg Lys Pro Ala Phe Leu
Ser Gly Glu Gln Lys Lys Ala 740 745 750 Ile Val Asp Leu Leu Phe Lys
Thr Asn Arg Lys Val Thr Val Lys Gln 755 760 765 Leu Lys Glu Asp Tyr
Phe Lys Lys Ile Glu Cys Phe Asp Ser Val Glu 770 775 780 Ile Ser Gly
Val Glu Asp Arg Phe Asn Ala Ser Leu Gly Thr Tyr His 785 790 795 800
Asp Leu Leu Lys Ile Ile Lys Asp Lys Asp Phe Leu Asp Asn Glu Glu 805
810 815 Asn Glu Asp Ile Leu Glu Asp Ile Val Leu Thr Leu Thr Leu Phe
Glu 820 825 830 Asp Arg Glu Met Ile Glu Glu Arg Leu Lys Thr Tyr Ala
His Leu Phe 835 840 845 Asp Asp Lys Val Met Lys Gln Leu Lys Arg Arg
Arg Tyr Thr Gly Trp 850 855 860 Gly Arg Leu Ser Arg Lys Leu Ile Asn
Gly Ile Arg Asp Lys Gln Ser 865 870 875 880 Gly Lys Thr Ile Leu Asp
Phe Leu Lys Ser Asp Gly Phe Ala Asn Arg 885 890 895 Asn Phe Met Gln
Leu Ile His Asp Asp Ser Leu Thr Phe Lys Glu Asp 900 905 910 Ile Gln
Lys Ala Gln Val Ser Gly Gln Gly Asp Ser Leu His Glu His 915 920 925
Ile Ala Asn Leu Ala Gly Ser Pro Ala Ile Lys Lys Gly Ile Leu Gln 930
935 940 Thr Val Lys Val Val Asp Glu Leu Val Lys Val Met Gly Arg His
Lys 945 950 955 960 Pro Glu Asn Ile Val Ile Glu Met Ala Arg Glu Asn
Gln Thr Thr Gln 965 970 975 Lys Gly Gln Lys Asn Ser Arg Glu Arg Met
Lys Arg Ile Glu Glu Gly 980 985 990 Ile Lys Glu Leu Gly Ser Gln Ile
Leu Lys Glu His Pro Val Glu Asn 995 1000 1005 Thr Gln Leu Gln Asn
Glu Lys Leu Tyr Leu Tyr Tyr Leu Gln Asn 1010 1015 1020 Gly Arg Asp
Met Tyr Val Asp Gln Glu Leu Asp Ile Asn Arg Leu 1025 1030 1035 Ser
Asp Tyr Asp Val Asp His Ile Val Pro Gln Ser Phe Leu Lys 1040 1045
1050 Asp Asp Ser Ile Asp Asn Lys Val Leu Thr Arg Ser Asp Lys Asn
1055 1060 1065 Arg Gly Lys Ser Asp Asn Val Pro Ser Glu Glu Val Val
Lys Lys 1070 1075 1080 Met Lys Asn Tyr Trp Arg Gln Leu Leu Asn Ala
Lys Leu Ile Thr 1085 1090 1095 Gln Arg Lys Phe Asp Asn Leu Thr Lys
Ala Glu Arg Gly Gly Leu 1100 1105 1110 Ser Glu Leu Asp Lys Ala Gly
Phe Ile Lys Arg Gln Leu Val Glu 1115 1120 1125 Thr Arg Gln Ile Thr
Lys His Val Ala Gln Ile Leu Asp Ser Arg 1130 1135 1140 Met Asn Thr
Lys Tyr Asp Glu Asn Asp Lys Leu Ile Arg Glu Val 1145 1150 1155 Lys
Val Ile Thr Leu Lys Ser Lys Leu Val Ser Asp Phe Arg Lys 1160 1165
1170 Asp Phe Gln Phe Tyr Lys Val Arg Glu Ile Asn Asn Tyr His His
1175 1180 1185 Ala His Asp Ala Tyr Leu Asn Ala Val Val Gly Thr Ala
Leu Ile 1190 1195 1200 Lys Lys Tyr Pro Lys Leu Glu Ser Glu Phe Val
Tyr Gly Asp Tyr 1205 1210 1215 Lys Val Tyr Asp Val Arg Lys Met Ile
Ala Lys Ser Glu Gln Glu 1220 1225 1230 Ile Gly Lys Ala Thr Ala Lys
Tyr Phe Phe Tyr Ser Asn Ile Met 1235 1240 1245 Asn Phe Phe Lys Thr
Glu Ile Thr Leu Ala Asn Gly Glu Ile Arg 1250 1255 1260 Lys Arg Pro
Leu Ile Glu Thr Asn Gly Glu Thr Gly Glu Ile Val 1265 1270 1275 Trp
Asp Lys Gly Arg Asp Phe Ala Thr Val Arg Lys Val Leu Ser 1280 1285
1290 Met Pro Gln Val Asn Ile Val Lys Lys Thr Glu Val Gln Thr Gly
1295 1300 1305 Gly Phe Ser Lys Glu Ser Ile Leu Pro Lys Arg Asn Ser
Asp Lys 1310 1315 1320 Leu Ile Ala Arg Lys Lys Asp Trp Asp Pro Lys
Lys Tyr Gly Gly 1325 1330 1335 Phe Asp Ser Pro Thr Val Ala Tyr Ser
Val Leu Val Val Ala Lys 1340 1345 1350 Val Glu Lys Gly Lys Ser Lys
Lys Leu Lys Ser Val Lys Glu Leu 1355 1360 1365 Leu Gly Ile Thr Ile
Met Glu Arg Ser Ser Phe Glu Lys Asn Pro 1370 1375 1380 Ile Asp Phe
Leu Glu Ala Lys Gly Tyr Lys Glu Val Lys Lys Asp 1385 1390 1395 Leu
Ile Ile Lys Leu Pro Lys Tyr Ser Leu Phe Glu Leu Glu Asn 1400 1405
1410 Gly Arg Lys Arg Met Leu Ala Ser Ala Gly Glu Leu Gln Lys Gly
1415 1420 1425 Asn Glu Leu Ala Leu Pro Ser Lys Tyr Val Asn Phe Leu
Tyr Leu 1430 1435 1440 Ala Ser His Tyr Glu Lys Leu Lys Gly Ser Pro
Glu Asp Asn Glu 1445 1450 1455 Gln Lys Gln Leu Phe Val Glu Gln His
Lys His Tyr Leu Asp Glu 1460 1465 1470 Ile Ile Glu Gln Ile Ser Glu
Phe Ser Lys Arg Val Ile Leu Ala 1475 1480 1485 Asp Ala Asn Leu Asp
Lys Val Leu Ser Ala Tyr Asn
Lys His Arg 1490 1495 1500 Asp Lys Pro Ile Arg Glu Gln Ala Glu Asn
Ile Ile His Leu Phe 1505 1510 1515 Thr Leu Thr Asn Leu Gly Ala Pro
Ala Ala Phe Lys Tyr Phe Asp 1520 1525 1530 Thr Thr Ile Asp Arg Lys
Arg Tyr Thr Ser Thr Lys Glu Val Leu 1535 1540 1545 Asp Ala Thr Leu
Ile His Gln Ser Ile Thr Gly Leu Tyr Glu Thr 1550 1555 1560 Arg Ile
Asp Leu Ser Gln Leu Gly Gly Asp Lys Arg Pro Ala Ala 1565 1570 1575
Thr Lys Lys Ala Gly Gln Ala Lys Lys Lys Lys 1580 1585
601583PRTArtificial Sequencesource/note="Description of Artificial
Sequence Synthetic polypeptide" 60Met Asp Tyr Lys Asp His Asp Gly
Asp Tyr Lys Asp His Asp Ile Asp 1 5 10 15 Tyr Lys Asp Asp Asp Asp
Lys Met Ala Pro Lys Lys Lys Arg Lys Val 20 25 30 Gly Ile His Gly
Val Pro Ala Ala Asp Lys Lys Tyr Ser Ile Gly Leu 35 40 45 Asp Ile
Gly Thr Asn Ser Val Gly Trp Ala Val Ile Thr Asp Glu Tyr 50 55 60
Lys Val Pro Ser Lys Lys Phe Lys Val Leu Gly Asn Thr Asp Arg His 65
70 75 80 Ser Ile Lys Lys Asn Leu Ile Gly Ala Leu Leu Phe Asp Ser
Gly Glu 85 90 95 Thr Ala Glu Ala Thr Arg Leu Lys Arg Thr Ala Arg
Arg Arg Tyr Thr 100 105 110 Arg Arg Lys Asn Arg Ile Cys Tyr Leu Gln
Glu Ile Phe Ser Asn Glu 115 120 125 Met Ala Lys Val Asp Asp Ser Phe
Phe His Arg Leu Glu Glu Ser Phe 130 135 140 Leu Val Glu Glu Asp Lys
Lys His Glu Arg His Pro Ile Phe Gly Asn 145 150 155 160 Ile Val Asp
Glu Val Ala Tyr His Glu Lys Tyr Pro Thr Ile Tyr His 165 170 175 Leu
Arg Lys Lys Leu Val Asp Ser Thr Asp Lys Ala Asp Leu Arg Leu 180 185
190 Ile Tyr Leu Ala Leu Ala His Met Ile Lys Phe Arg Gly His Phe Leu
195 200 205 Ile Glu Gly Asp Leu Asn Pro Asp Asn Ser Asp Val Asp Lys
Leu Phe 210 215 220 Ile Gln Leu Val Gln Thr Tyr Asn Gln Leu Phe Glu
Glu Asn Pro Ile 225 230 235 240 Asn Ala Ser Gly Val Asp Ala Lys Ala
Ile Leu Ser Ala Arg Leu Ser 245 250 255 Lys Ser Arg Arg Leu Glu Asn
Leu Ile Ala Gln Leu Pro Gly Glu Lys 260 265 270 Lys Asn Gly Leu Phe
Gly Asn Leu Ile Ala Leu Ser Leu Gly Leu Thr 275 280 285 Pro Asn Phe
Lys Ser Asn Phe Asp Leu Ala Glu Asp Ala Lys Leu Gln 290 295 300 Leu
Ser Lys Asp Thr Tyr Asp Asp Asp Leu Asp Asn Leu Leu Ala Gln 305 310
315 320 Ile Gly Asp Gln Tyr Ala Asp Leu Phe Leu Ala Ala Lys Asn Leu
Ser 325 330 335 Asp Ala Ile Leu Leu Ser Asp Ile Leu Arg Val Asn Thr
Glu Ile Thr 340 345 350 Lys Ala Pro Leu Ser Ala Ser Met Ile Lys Arg
Tyr Asp Glu His His 355 360 365 Gln Asp Leu Thr Leu Leu Lys Ala Leu
Val Arg Gln Gln Leu Pro Glu 370 375 380 Lys Tyr Lys Glu Ile Phe Phe
Asp Gln Ser Lys Asn Gly Tyr Ala Gly 385 390 395 400 Tyr Ile Asp Gly
Gly Ala Ser Gln Glu Glu Phe Tyr Lys Phe Ile Lys 405 410 415 Pro Ile
Leu Glu Lys Met Asp Gly Thr Glu Glu Leu Leu Val Lys Leu 420 425 430
Asn Arg Glu Asp Leu Leu Arg Lys Gln Arg Thr Phe Asp Asn Gly Ser 435
440 445 Ile Pro His Gln Ile His Leu Gly Glu Leu His Ala Ile Leu Arg
Arg 450 455 460 Gln Glu Asp Phe Tyr Pro Phe Leu Lys Asp Asn Arg Glu
Lys Ile Glu 465 470 475 480 Lys Ile Leu Thr Phe Arg Ile Pro Tyr Tyr
Val Gly Pro Leu Ala Arg 485 490 495 Gly Asn Ser Arg Phe Ala Trp Met
Thr Arg Lys Ser Glu Glu Thr Ile 500 505 510 Thr Pro Trp Asn Phe Glu
Glu Val Val Asp Lys Gly Ala Ser Ala Gln 515 520 525 Ser Phe Ile Glu
Arg Met Thr Asn Phe Asp Lys Asn Leu Pro Asn Glu 530 535 540 Lys Val
Leu Pro Lys His Ser Leu Leu Tyr Glu Tyr Phe Thr Val Tyr 545 550 555
560 Asn Glu Leu Thr Lys Val Lys Tyr Val Thr Glu Gly Met Arg Lys Pro
565 570 575 Ala Phe Leu Ser Gly Glu Gln Lys Lys Ala Ile Val Asp Leu
Leu Phe 580 585 590 Lys Thr Asn Arg Lys Val Thr Val Lys Gln Leu Lys
Glu Asp Tyr Phe 595 600 605 Lys Lys Ile Glu Cys Phe Asp Ser Val Glu
Ile Ser Gly Val Glu Asp 610 615 620 Arg Phe Asn Ala Ser Leu Gly Thr
Tyr His Asp Leu Leu Lys Ile Ile 625 630 635 640 Lys Asp Lys Asp Phe
Leu Asp Asn Glu Glu Asn Glu Asp Ile Leu Glu 645 650 655 Asp Ile Val
Leu Thr Leu Thr Leu Phe Glu Asp Arg Glu Met Ile Glu 660 665 670 Glu
Arg Leu Lys Thr Tyr Ala His Leu Phe Asp Asp Lys Val Met Lys 675 680
685 Gln Leu Lys Arg Arg Arg Tyr Thr Gly Trp Gly Arg Leu Ser Arg Lys
690 695 700 Leu Ile Asn Gly Ile Arg Asp Lys Gln Ser Gly Lys Thr Ile
Leu Asp 705 710 715 720 Phe Leu Lys Ser Asp Gly Phe Ala Asn Arg Asn
Phe Met Gln Leu Ile 725 730 735 His Asp Asp Ser Leu Thr Phe Lys Glu
Asp Ile Gln Lys Ala Gln Val 740 745 750 Ser Gly Gln Gly Asp Ser Leu
His Glu His Ile Ala Asn Leu Ala Gly 755 760 765 Ser Pro Ala Ile Lys
Lys Gly Ile Leu Gln Thr Val Lys Val Val Asp 770 775 780 Glu Leu Val
Lys Val Met Gly Arg His Lys Pro Glu Asn Ile Val Ile 785 790 795 800
Glu Met Ala Arg Glu Asn Gln Thr Thr Gln Lys Gly Gln Lys Asn Ser 805
810 815 Arg Glu Arg Met Lys Arg Ile Glu Glu Gly Ile Lys Glu Leu Gly
Ser 820 825 830 Gln Ile Leu Lys Glu His Pro Val Glu Asn Thr Gln Leu
Gln Asn Glu 835 840 845 Lys Leu Tyr Leu Tyr Tyr Leu Gln Asn Gly Arg
Asp Met Tyr Val Asp 850 855 860 Gln Glu Leu Asp Ile Asn Arg Leu Ser
Asp Tyr Asp Val Asp His Ile 865 870 875 880 Val Pro Gln Ser Phe Leu
Lys Asp Asp Ser Ile Asp Asn Lys Val Leu 885 890 895 Thr Arg Ser Asp
Lys Asn Arg Gly Lys Ser Asp Asn Val Pro Ser Glu 900 905 910 Glu Val
Val Lys Lys Met Lys Asn Tyr Trp Arg Gln Leu Leu Asn Ala 915 920 925
Lys Leu Ile Thr Gln Arg Lys Phe Asp Asn Leu Thr Lys Ala Glu Arg 930
935 940 Gly Gly Leu Ser Glu Leu Asp Lys Ala Gly Phe Ile Lys Arg Gln
Leu 945 950 955 960 Val Glu Thr Arg Gln Ile Thr Lys His Val Ala Gln
Ile Leu Asp Ser 965 970 975 Arg Met Asn Thr Lys Tyr Asp Glu Asn Asp
Lys Leu Ile Arg Glu Val 980 985 990 Lys Val Ile Thr Leu Lys Ser Lys
Leu Val Ser Asp Phe Arg Lys Asp 995 1000 1005 Phe Gln Phe Tyr Lys
Val Arg Glu Ile Asn Asn Tyr His His Ala 1010 1015 1020 His Asp Ala
Tyr Leu Asn Ala Val Val Gly Thr Ala Leu Ile Lys 1025 1030 1035 Lys
Tyr Pro Lys Leu Glu Ser Glu Phe Val Tyr Gly Asp Tyr Lys 1040 1045
1050 Val Tyr Asp Val Arg Lys Met Ile Ala Lys Ser Glu Gln Glu Ile
1055 1060 1065 Gly Lys Ala Thr Ala Lys Tyr Phe Phe Tyr Ser Asn Ile
Met Asn 1070 1075 1080 Phe Phe Lys Thr Glu Ile Thr Leu Ala Asn Gly
Glu Ile Arg Lys 1085 1090 1095 Arg Pro Leu Ile Glu Thr Asn Gly Glu
Thr Gly Glu Ile Val Trp 1100 1105 1110 Asp Lys Gly Arg Asp Phe Ala
Thr Val Arg Lys Val Leu Ser Met 1115 1120 1125 Pro Gln Val Asn Ile
Val Lys Lys Thr Glu Val Gln Thr Gly Gly 1130 1135 1140 Phe Ser Lys
Glu Ser Ile Leu Pro Lys Arg Asn Ser Asp Lys Leu 1145 1150 1155 Ile
Ala Arg Lys Lys Asp Trp Asp Pro Lys Lys Tyr Gly Gly Phe 1160 1165
1170 Asp Ser Pro Thr Val Ala Tyr Ser Val Leu Val Val Ala Lys Val
1175 1180 1185 Glu Lys Gly Lys Ser Lys Lys Leu Lys Ser Val Lys Glu
Leu Leu 1190 1195 1200 Gly Ile Thr Ile Met Glu Arg Ser Ser Phe Glu
Lys Asn Pro Ile 1205 1210 1215 Asp Phe Leu Glu Ala Lys Gly Tyr Lys
Glu Val Lys Lys Asp Leu 1220 1225 1230 Ile Ile Lys Leu Pro Lys Tyr
Ser Leu Phe Glu Leu Glu Asn Gly 1235 1240 1245 Arg Lys Arg Met Leu
Ala Ser Ala Gly Glu Leu Gln Lys Gly Asn 1250 1255 1260 Glu Leu Ala
Leu Pro Ser Lys Tyr Val Asn Phe Leu Tyr Leu Ala 1265 1270 1275 Ser
His Tyr Glu Lys Leu Lys Gly Ser Pro Glu Asp Asn Glu Gln 1280 1285
1290 Lys Gln Leu Phe Val Glu Gln His Lys His Tyr Leu Asp Glu Ile
1295 1300 1305 Ile Glu Gln Ile Ser Glu Phe Ser Lys Arg Val Ile Leu
Ala Asp 1310 1315 1320 Ala Asn Leu Asp Lys Val Leu Ser Ala Tyr Asn
Lys His Arg Asp 1325 1330 1335 Lys Pro Ile Arg Glu Gln Ala Glu Asn
Ile Ile His Leu Phe Thr 1340 1345 1350 Leu Thr Asn Leu Gly Ala Pro
Ala Ala Phe Lys Tyr Phe Asp Thr 1355 1360 1365 Thr Ile Asp Arg Lys
Arg Tyr Thr Ser Thr Lys Glu Val Leu Asp 1370 1375 1380 Ala Thr Leu
Ile His Gln Ser Ile Thr Gly Leu Tyr Glu Thr Arg 1385 1390 1395 Ile
Asp Leu Ser Gln Leu Gly Gly Asp Lys Arg Pro Ala Ala Thr 1400 1405
1410 Lys Lys Ala Gly Gln Ala Lys Lys Lys Lys Gly Ser Ile Ser Leu
1415 1420 1425 Ile Ala Ala Leu Ala Val Asp His Val Ile Gly Met Glu
Thr Val 1430 1435 1440 Met Pro Trp Asn Leu Pro Ala Asp Leu Ala Trp
Phe Lys Arg Asn 1445 1450 1455 Thr Leu Asn Lys Pro Val Ile Met Gly
Arg His Thr Trp Glu Ser 1460 1465 1470 Ile Gly Arg Pro Leu Pro Gly
Arg Lys Asn Ile Ile Leu Ser Ser 1475 1480 1485 Gln Pro Ser Thr Asp
Asp Arg Val Thr Trp Val Lys Ser Val Asp 1490 1495 1500 Glu Ala Ile
Ala Ala Cys Gly Asp Val Pro Glu Ile Met Val Ile 1505 1510 1515 Gly
Gly Gly Arg Val Tyr Glu Gln Phe Leu Pro Lys Ala Gln Lys 1520 1525
1530 Leu Tyr Leu Thr His Ile Asp Ala Glu Val Glu Gly Asp Thr His
1535 1540 1545 Phe Pro Asp Tyr Glu Pro Asp Asp Trp Glu Ser Val Phe
Ser Glu 1550 1555 1560 Phe His Asp Ala Asp Ala Gln Asn Ser His Ser
Tyr Cys Phe Glu 1565 1570 1575 Ile Leu Glu Arg Arg 1580
611747PRTArtificial Sequencesource/note="Description of Artificial
Sequence Synthetic polypeptide" 61Met Asp Tyr Lys Asp His Asp Gly
Asp Tyr Lys Asp His Asp Ile Asp 1 5 10 15 Tyr Lys Asp Asp Asp Asp
Lys Met Ala Pro Lys Lys Lys Arg Lys Val 20 25 30 Gly Ile His Gly
Val Pro Ala Ala Gly Ser Ile Ser Leu Ile Ala Ala 35 40 45 Leu Ala
Val Asp Tyr Val Ile Gly Met Glu Asn Ala Met Pro Trp Asn 50 55 60
Leu Pro Ala Asp Leu Ala Trp Phe Lys Arg Asn Thr Leu Asn Lys Pro 65
70 75 80 Val Ile Met Gly Arg His Thr Trp Glu Ser Ile Gly Arg Pro
Leu Pro 85 90 95 Gly Arg Lys Asn Ile Ile Leu Ser Ser Gln Pro Ser
Thr Asp Asp Arg 100 105 110 Val Thr Trp Val Lys Ser Val Asp Glu Ala
Ile Ala Ala Cys Gly Asp 115 120 125 Val Pro Glu Ile Met Val Ile Gly
Gly Gly Arg Val Ile Glu Gln Phe 130 135 140 Leu Pro Lys Ala Gln Lys
Leu Tyr Leu Thr His Ile Asp Ala Glu Val 145 150 155 160 Glu Gly Asp
Thr His Phe Pro Asp Tyr Glu Pro Asp Asp Trp Glu Ser 165 170 175 Val
Phe Ser Glu Phe His Asp Ala Asp Ala Gln Asn Ser His Ser Tyr 180 185
190 Cys Phe Glu Ile Leu Glu Arg Arg Gly Ser Gly Ser Gly Ser Asp Lys
195 200 205 Lys Tyr Ser Ile Gly Leu Asp Ile Gly Thr Asn Ser Val Gly
Trp Ala 210 215 220 Val Ile Thr Asp Glu Tyr Lys Val Pro Ser Lys Lys
Phe Lys Val Leu 225 230 235 240 Gly Asn Thr Asp Arg His Ser Ile Lys
Lys Asn Leu Ile Gly Ala Leu 245 250 255 Leu Phe Asp Ser Gly Glu Thr
Ala Glu Ala Thr Arg Leu Lys Arg Thr 260 265 270 Ala Arg Arg Arg Tyr
Thr Arg Arg Lys Asn Arg Ile Cys Tyr Leu Gln 275 280 285 Glu Ile Phe
Ser Asn Glu Met Ala Lys Val Asp Asp Ser Phe Phe His 290 295 300 Arg
Leu Glu Glu Ser Phe Leu Val Glu Glu Asp Lys Lys His Glu Arg 305 310
315 320 His Pro Ile Phe Gly Asn Ile Val Asp Glu Val Ala Tyr His Glu
Lys 325 330 335 Tyr Pro Thr Ile Tyr His Leu Arg Lys Lys Leu Val Asp
Ser Thr Asp 340 345 350 Lys Ala Asp Leu Arg Leu Ile Tyr Leu Ala Leu
Ala His Met Ile Lys 355 360 365 Phe Arg Gly His Phe Leu Ile Glu Gly
Asp Leu Asn Pro Asp Asn Ser 370 375 380 Asp Val Asp Lys Leu Phe Ile
Gln Leu Val Gln Thr Tyr Asn Gln Leu 385 390 395 400 Phe Glu Glu Asn
Pro Ile Asn Ala Ser Gly Val Asp Ala Lys Ala Ile 405 410 415 Leu Ser
Ala Arg Leu Ser Lys Ser Arg Arg Leu Glu Asn Leu Ile Ala 420 425 430
Gln Leu Pro Gly Glu Lys Lys Asn Gly Leu Phe Gly Asn Leu Ile Ala 435
440 445 Leu Ser Leu Gly Leu Thr Pro Asn Phe Lys Ser Asn Phe Asp Leu
Ala 450 455 460 Glu Asp Ala Lys Leu Gln Leu Ser Lys Asp Thr Tyr Asp
Asp Asp Leu 465 470 475 480 Asp Asn Leu Leu Ala Gln Ile Gly Asp Gln
Tyr Ala Asp Leu Phe Leu 485 490 495 Ala Ala Lys Asn Leu Ser Asp Ala
Ile Leu Leu Ser Asp Ile Leu Arg 500 505 510 Val Asn Thr Glu Ile Thr
Lys Ala Pro Leu Ser Ala Ser Met Ile Lys 515 520 525 Arg Tyr Asp Glu
His His Gln Asp Leu Thr Leu Leu Lys Ala Leu Val 530 535 540 Arg Gln
Gln Leu Pro Glu Lys Tyr Lys Glu Ile Phe Phe Asp Gln Ser 545 550 555
560 Lys Asn Gly Tyr Ala Gly Tyr Ile Asp
Gly Gly Ala Ser Gln Glu Glu 565 570 575 Phe Tyr Lys Phe Ile Lys Pro
Ile Leu Glu Lys Met Asp Gly Thr Glu 580 585 590 Glu Leu Leu Val Lys
Leu Asn Arg Glu Asp Leu Leu Arg Lys Gln Arg 595 600 605 Thr Phe Asp
Asn Gly Ser Ile Pro His Gln Ile His Leu Gly Glu Leu 610 615 620 His
Ala Ile Leu Arg Arg Gln Glu Asp Phe Tyr Pro Phe Leu Lys Asp 625 630
635 640 Asn Arg Glu Lys Ile Glu Lys Ile Leu Thr Phe Arg Ile Pro Tyr
Tyr 645 650 655 Val Gly Pro Leu Ala Arg Gly Asn Ser Arg Phe Ala Trp
Met Thr Arg 660 665 670 Lys Ser Glu Glu Thr Ile Thr Pro Trp Asn Phe
Glu Glu Val Val Asp 675 680 685 Lys Gly Ala Ser Ala Gln Ser Phe Ile
Glu Arg Met Thr Asn Phe Asp 690 695 700 Lys Asn Leu Pro Asn Glu Lys
Val Leu Pro Lys His Ser Leu Leu Tyr 705 710 715 720 Glu Tyr Phe Thr
Val Tyr Asn Glu Leu Thr Lys Val Lys Tyr Val Thr 725 730 735 Glu Gly
Met Arg Lys Pro Ala Phe Leu Ser Gly Glu Gln Lys Lys Ala 740 745 750
Ile Val Asp Leu Leu Phe Lys Thr Asn Arg Lys Val Thr Val Lys Gln 755
760 765 Leu Lys Glu Asp Tyr Phe Lys Lys Ile Glu Cys Phe Asp Ser Val
Glu 770 775 780 Ile Ser Gly Val Glu Asp Arg Phe Asn Ala Ser Leu Gly
Thr Tyr His 785 790 795 800 Asp Leu Leu Lys Ile Ile Lys Asp Lys Asp
Phe Leu Asp Asn Glu Glu 805 810 815 Asn Glu Asp Ile Leu Glu Asp Ile
Val Leu Thr Leu Thr Leu Phe Glu 820 825 830 Asp Arg Glu Met Ile Glu
Glu Arg Leu Lys Thr Tyr Ala His Leu Phe 835 840 845 Asp Asp Lys Val
Met Lys Gln Leu Lys Arg Arg Arg Tyr Thr Gly Trp 850 855 860 Gly Arg
Leu Ser Arg Lys Leu Ile Asn Gly Ile Arg Asp Lys Gln Ser 865 870 875
880 Gly Lys Thr Ile Leu Asp Phe Leu Lys Ser Asp Gly Phe Ala Asn Arg
885 890 895 Asn Phe Met Gln Leu Ile His Asp Asp Ser Leu Thr Phe Lys
Glu Asp 900 905 910 Ile Gln Lys Ala Gln Val Ser Gly Gln Gly Asp Ser
Leu His Glu His 915 920 925 Ile Ala Asn Leu Ala Gly Ser Pro Ala Ile
Lys Lys Gly Ile Leu Gln 930 935 940 Thr Val Lys Val Val Asp Glu Leu
Val Lys Val Met Gly Arg His Lys 945 950 955 960 Pro Glu Asn Ile Val
Ile Glu Met Ala Arg Glu Asn Gln Thr Thr Gln 965 970 975 Lys Gly Gln
Lys Asn Ser Arg Glu Arg Met Lys Arg Ile Glu Glu Gly 980 985 990 Ile
Lys Glu Leu Gly Ser Gln Ile Leu Lys Glu His Pro Val Glu Asn 995
1000 1005 Thr Gln Leu Gln Asn Glu Lys Leu Tyr Leu Tyr Tyr Leu Gln
Asn 1010 1015 1020 Gly Arg Asp Met Tyr Val Asp Gln Glu Leu Asp Ile
Asn Arg Leu 1025 1030 1035 Ser Asp Tyr Asp Val Asp His Ile Val Pro
Gln Ser Phe Leu Lys 1040 1045 1050 Asp Asp Ser Ile Asp Asn Lys Val
Leu Thr Arg Ser Asp Lys Asn 1055 1060 1065 Arg Gly Lys Ser Asp Asn
Val Pro Ser Glu Glu Val Val Lys Lys 1070 1075 1080 Met Lys Asn Tyr
Trp Arg Gln Leu Leu Asn Ala Lys Leu Ile Thr 1085 1090 1095 Gln Arg
Lys Phe Asp Asn Leu Thr Lys Ala Glu Arg Gly Gly Leu 1100 1105 1110
Ser Glu Leu Asp Lys Ala Gly Phe Ile Lys Arg Gln Leu Val Glu 1115
1120 1125 Thr Arg Gln Ile Thr Lys His Val Ala Gln Ile Leu Asp Ser
Arg 1130 1135 1140 Met Asn Thr Lys Tyr Asp Glu Asn Asp Lys Leu Ile
Arg Glu Val 1145 1150 1155 Lys Val Ile Thr Leu Lys Ser Lys Leu Val
Ser Asp Phe Arg Lys 1160 1165 1170 Asp Phe Gln Phe Tyr Lys Val Arg
Glu Ile Asn Asn Tyr His His 1175 1180 1185 Ala His Asp Ala Tyr Leu
Asn Ala Val Val Gly Thr Ala Leu Ile 1190 1195 1200 Lys Lys Tyr Pro
Lys Leu Glu Ser Glu Phe Val Tyr Gly Asp Tyr 1205 1210 1215 Lys Val
Tyr Asp Val Arg Lys Met Ile Ala Lys Ser Glu Gln Glu 1220 1225 1230
Ile Gly Lys Ala Thr Ala Lys Tyr Phe Phe Tyr Ser Asn Ile Met 1235
1240 1245 Asn Phe Phe Lys Thr Glu Ile Thr Leu Ala Asn Gly Glu Ile
Arg 1250 1255 1260 Lys Arg Pro Leu Ile Glu Thr Asn Gly Glu Thr Gly
Glu Ile Val 1265 1270 1275 Trp Asp Lys Gly Arg Asp Phe Ala Thr Val
Arg Lys Val Leu Ser 1280 1285 1290 Met Pro Gln Val Asn Ile Val Lys
Lys Thr Glu Val Gln Thr Gly 1295 1300 1305 Gly Phe Ser Lys Glu Ser
Ile Leu Pro Lys Arg Asn Ser Asp Lys 1310 1315 1320 Leu Ile Ala Arg
Lys Lys Asp Trp Asp Pro Lys Lys Tyr Gly Gly 1325 1330 1335 Phe Asp
Ser Pro Thr Val Ala Tyr Ser Val Leu Val Val Ala Lys 1340 1345 1350
Val Glu Lys Gly Lys Ser Lys Lys Leu Lys Ser Val Lys Glu Leu 1355
1360 1365 Leu Gly Ile Thr Ile Met Glu Arg Ser Ser Phe Glu Lys Asn
Pro 1370 1375 1380 Ile Asp Phe Leu Glu Ala Lys Gly Tyr Lys Glu Val
Lys Lys Asp 1385 1390 1395 Leu Ile Ile Lys Leu Pro Lys Tyr Ser Leu
Phe Glu Leu Glu Asn 1400 1405 1410 Gly Arg Lys Arg Met Leu Ala Ser
Ala Gly Glu Leu Gln Lys Gly 1415 1420 1425 Asn Glu Leu Ala Leu Pro
Ser Lys Tyr Val Asn Phe Leu Tyr Leu 1430 1435 1440 Ala Ser His Tyr
Glu Lys Leu Lys Gly Ser Pro Glu Asp Asn Glu 1445 1450 1455 Gln Lys
Gln Leu Phe Val Glu Gln His Lys His Tyr Leu Asp Glu 1460 1465 1470
Ile Ile Glu Gln Ile Ser Glu Phe Ser Lys Arg Val Ile Leu Ala 1475
1480 1485 Asp Ala Asn Leu Asp Lys Val Leu Ser Ala Tyr Asn Lys His
Arg 1490 1495 1500 Asp Lys Pro Ile Arg Glu Gln Ala Glu Asn Ile Ile
His Leu Phe 1505 1510 1515 Thr Leu Thr Asn Leu Gly Ala Pro Ala Ala
Phe Lys Tyr Phe Asp 1520 1525 1530 Thr Thr Ile Asp Arg Lys Arg Tyr
Thr Ser Thr Lys Glu Val Leu 1535 1540 1545 Asp Ala Thr Leu Ile His
Gln Ser Ile Thr Gly Leu Tyr Glu Thr 1550 1555 1560 Arg Ile Asp Leu
Ser Gln Leu Gly Gly Asp Lys Arg Pro Ala Ala 1565 1570 1575 Thr Lys
Lys Ala Gly Gln Ala Lys Lys Lys Lys Ile Ser Leu Ile 1580 1585 1590
Ala Ala Leu Ala Val Asp His Val Ile Gly Met Glu Thr Val Met 1595
1600 1605 Pro Trp Asn Leu Pro Ala Asp Leu Ala Trp Phe Lys Arg Asn
Thr 1610 1615 1620 Leu Asn Lys Pro Val Ile Met Gly Arg His Thr Trp
Glu Ser Ile 1625 1630 1635 Gly Arg Pro Leu Pro Gly Arg Lys Asn Ile
Ile Leu Ser Ser Gln 1640 1645 1650 Pro Ser Thr Asp Asp Arg Val Thr
Trp Val Lys Ser Val Asp Glu 1655 1660 1665 Ala Ile Ala Ala Cys Gly
Asp Val Pro Glu Ile Met Val Ile Gly 1670 1675 1680 Gly Gly Arg Val
Tyr Glu Gln Phe Leu Pro Lys Ala Gln Lys Leu 1685 1690 1695 Tyr Leu
Thr His Ile Asp Ala Glu Val Glu Gly Asp Thr His Phe 1700 1705 1710
Pro Asp Tyr Glu Pro Asp Asp Trp Glu Ser Val Phe Ser Glu Phe 1715
1720 1725 His Asp Ala Asp Ala Gln Asn Ser His Ser Tyr Cys Phe Glu
Ile 1730 1735 1740 Leu Glu Arg Arg 1745 6223DNAArtificial
Sequencesource/note="Description of Artificial Sequence Synthetic
oligonucleotide"modified_base(1)..(20)a, c, t or
gmodified_base(21)..(21)a, c, t, g, unknown or other 62nnnnnnnnnn
nnnnnnnnnn ngg 236315DNAArtificial Sequencesource/note="Description
of Artificial Sequence Synthetic
oligonucleotide"modified_base(1)..(12)a, c, t or
gmodified_base(13)..(13)a, c, t, g, unknown or other 63nnnnnnnnnn
nnngg 156423DNAArtificial Sequencesource/note="Description of
Artificial Sequence Synthetic
oligonucleotide"modified_base(1)..(20)a, c, t or
gmodified_base(21)..(21)a, c, t, g, unknown or other 64nnnnnnnnnn
nnnnnnnnnn ngg 236514DNAArtificial Sequencesource/note="Description
of Artificial Sequence Synthetic
oligonucleotide"modified_base(1)..(11)a, c, t or
gmodified_base(12)..(12)a, c, t, g, unknown or other 65nnnnnnnnnn
nngg 146625DNAArtificial Sequencesource/note="Description of
Artificial Sequence Synthetic
oligonucleotide"modified_base(1)..(20)a, c, t or
gmodified_base(21)..(21)a, c, t, g, unknown or
othermodified_base(24)..(24)a, c, t, g, unknown or other
66nnnnnnnnnn nnnnnnnnnn nggng 256717DNAArtificial
Sequencesource/note="Description of Artificial Sequence Synthetic
oligonucleotide"modified_base(1)..(12)a, c, t or
gmodified_base(13)..(13)a, c, t, g, unknown or
othermodified_base(16)..(16)a, c, t, g, unknown or other
67nnnnnnnnnn nnnggng 176825DNAArtificial
Sequencesource/note="Description of Artificial Sequence Synthetic
oligonucleotide"modified_base(1)..(20)a, c, t or
gmodified_base(21)..(21)a, c, t, g, unknown or
othermodified_base(24)..(24)a, c, t, g, unknown or other
68nnnnnnnnnn nnnnnnnnnn nggng 256916DNAArtificial
Sequencesource/note="Description of Artificial Sequence Synthetic
oligonucleotide"modified_base(1)..(11)a, c, t or
gmodified_base(12)..(12)a, c, t, g, unknown or
othermodified_base(15)..(15)a, c, t, g, unknown or other
69nnnnnnnnnn nnggng 16
* * * * *
References